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EB1, a microtubule tip-associated protein
To fulfil the requirement of the course of Reading and
analysis of structural biology

Submitted By:
Saima Akram
Student ID: BL1400802
Training Course: Ph.D
School of Life Sciences,
University of Science and Technology of China.

Submitted To:
Prof. Yuxing CHEN
Course Name: Reading and analysis of structural biology
Course ID: BI6620101

The faithful chromosome segregation during mitosis is orchestrated by dynamic
interaction between spindle microtubules and the kinetochore, a protein
supercomplex assembled at the chromosome centromere. Kinetochore acts as a
structural platform for linking the centromere to spindle microtubules and
functions as a signaling hub coordinating chromosome attachment and the
metaphase to anaphase transition (Welburn et al., 2008). The spindle assembly
checkpoint senses kinetochore-microtubule attachment and/or tension at the
kinetochore and delays the onset of anaphase until all chromosomes have
successfully achieved stable, bi-oriented attachments (Musacchio et al., 2007). In
the past decade, molecular mechanism of dynamic kinetochore-microtubule
interactions has achieved significant progress (Cheeseman et al., 2008). Multiple
proteins function together at the kinetochore; they contribute to the establishment
and/or regulation of dynamic kinetochore-microtubule interactions by forming
attachment sites for spindle microtubules. The kinetochore is a complex molecular
machine to power chromosome movements and orchestrates the checkpoint
signaling during cell division cycle progression. Proper alignment and segregation
of sister chromatids during mitosis is the most important event in eukaryotic cells;
it depends on the dynamic interaction between kinetochore and spindle
microtubules. To build effective kinetochore-microtubule attachment, it must
satisfy two criteria: one is to maintain the dynamic flux of kinetochore
microtubules, and the other is to secure the dynamic link between kinetochore and
microtubules. Numerous microtubule-associated proteins play very important roles

Eb1 (End binding 1 protein)
EB1 is a microtubule tip-associated protein that localizes at both the growing plusends of microtubules and the centrosome (Vaughan, 2005; Morrison, 2007). As an
important regulator of microtubule dynamics, EB1 is critically involved in many
microtubule-mediated cell activities, such as establishment and maintenance of cell
polarity, search and capture of chromosomes during mitosis, and positioning of the
mitotic spindle during asymmetric cell divisions (Morrison, 2007; Gundersen,
2002). EB1 was originally identified as a protein interacting with the adenomatous
polyposis coli (APC) tumor suppressor protein (Su LK, et al., 1995). The EB1–
APC complex has been shown to increase microtubule polymerization and
stabilize microtubule–kinetochore interactions in mitosis (Nakamura, et al., 2001).
In addition, the interaction of EB1 with APC appears to be required for efficient
cell migration (Wen Y, et al., 2004). In addition to its role in regulating
microtubule dynamics and microtubule-based cellular processes, EB1 has recently
been implicated in tumorigenesis. Proteomic studies using two-dimensional gel
electrophoresis identified EB1 over expression in human gastric carcinoma and
hepato cellular carcinoma (Nishigaki R, et al., 2005; Fujii K, et al., 2005). EB1 is
well known as a microtubule tip-associated protein. It functions together with the
APC tumor suppressor and p150 glued to modulate microtubule dynamics and is
involved in a variety of microtubule-based cellular processes (Vaughan, 2005).
Recent studies have shown that EB1 promotes cancer cell proliferation and have
implicated EB1 in tumorigenesis (Wang Y, et al., 2005), but the underlying
molecular mechanisms remain elusive. EB1 promotes the kinase activity of
Aurora-B. This is supported by the following evidence: (i) EB1 interacts
specifically with Aurora-B both in cells and in vitro; (ii) over expression of EB1
significantly enhances Aurora-B activity; (iii) knockdown of EB1 expression

. 1997). however. called BIM1 (binding to microtubules 1). Genome sequencing projects have revealed that budding yeast possess a single EB1 sequence homologue. However. 2005). EB1 is found in a similar distribution.. 2003). In the cells of higher organisms. because the protein interacted with α-tubulin in a two-hybrid screen (Schwartz et al. It should be noted. Adams et al. Eb1 Proteins Regulate Microtubule Dynamics. and Chromosome Stability The EB1 family represents a highly conserved group of proteins. the three Aurora kinases have quite different subcellular localizations and functions. 2003). and (iv) EB1 protects Aurora-B from inactivation by PP2A. 2003). especially at their distal tips. By indirect immunofluorescence in tissue culture .. lymphocytic. Cell Polarity. Aurora-B is important for chromosome segregation and cytokinesis (Carmena et al. on a more spatially extensive microtubule array. Given that Aurora-B activity is critical for cell proliferation and that increased Aurora-B activity is frequently detected in cancer (Katayama et al.diminishes Aurora-B activity substantially. present in yeast through humans that localize to spindle and cytoplasmic microtubules.. that in the cell there is a pool of soluble EB1 not associated with microtubules (Vaughan. Mammalian cells have three Aurora kinases that present similar structures (Carmena et al. 2001).. As a component of the chromosome passenger complex. This protein localizes at different places in the mitotic apparatus depending on the stage of mitosis (Carmena et al. EB1 has been found in every organism and nearly every cell type examined.. including neuronal. 2003. Aurora-B co localizes with EB1 on the central spindle in anaphase and in the mid body during cytokinesis. The full diversity of EB1 family members remains unknown at this time. It is not impossible that a portion of soluble EB1 may interact with Aurora-B at other cellular sites or in other mitotic phases. To date. and epithelial cells.

1997). end binding 1 aptly labels the protein. Thus. it promotes net polymerization by increasing both the time spent growing and the rescue frequency. 1998. In bim1Δ cells.cells. In wild-type budding yeast. Yeast lacking the BIM1 gene are viable. even though Bim1p increases the depolymerization rate. than in wild-type cells (Tirnauer et al. and the distal tips of cytoplasmic microtubules (Berrueta et al. what do EB1 proteins do to microtubules? The first studies of EB1 function have come from yeast mutants. possibly at or near kinetochores (Juwana et al. EB1 staining also have been visualized at what appear to be microtubule ends within the mitotic spindle.... Once bound. a location ideally suited for linking the microtubule cytoskeleton to other structures within the cell.. 1998). at the expense of growing. Although EB1 was named before its end-binding properties were known. 1999). but their cytoplasmic microtubules are shorter than those in wild-type cells and they show abnormalities in parameters of dynamic instability. 1999). the mitotic spindle. Thus. . which is the phase when the spindle pole body moves toward the presumptive bud site in preparation for mitosis (Carminati and Stearns. microtubules were found to depolymerize more slowly and to undergo fewer transitions and longer pauses. EB1 has been localized to the centrosome. microtubules are most dynamic during the G1 phase of the cell cycle. resulting in microtubules that are longer as well as more dynamic. Morrison et al. EB1 proteins are found on microtubule plus ends throughout the cell cycle in diverse cell types.

2 Ribbons diagrams of EB1 (left). Fig. It interacts with the carboxy terminus of the adenomatous polyposis coli (APC) tumor suppressor. Another binding partner of the EB1-C domain is the well conserved +TIPS protein dynactin. and least squares fit of the two structures with Bim1p shown exclusively in grey. a component of the large cytoplasmic dynein/dynactin complex. The EB1-C domain comprises a unique EB1-like sequence motif that acts as a binding site for other +TIP proteins. a well conserved +TIP phosphoprotein with a pivotal function in cell cycle regulation. Bim1p (center).1 EB1 binding domains EB1 members have a bipartite composition: the N-terminal CH domain mediates microtubule plus end localization and a C-terminal cargo binding domain (EB1-C) that captures cell polarity determinants.Structure of EB1 Fig. .

 Fission yeast microtubule integrity protein mal3. Some proteins known to contain an EB1-C domain are listed below:  Yeast protein BIM1. The two parallel alpha1 helices of the EB1-C domain dimer wrap around each other in a slightly left-handed supercoil. The domain is comprised of eight helices. two helical segments from each monomer form a four-helix bundle.The ~80-residue EB1-C domain starts with a long smoothly curved helix (alpha1). Specific differences between actin binding calponin . Structural determination of Nterminal domains from human EB1 and the EB1 homolog in Saccharomyces cerevisiae (Bim1p) reveals that the domain is indeed of the calponin homology fold. which is followed by a hairpin connection leading to a short second helix (alpha2) running antiparallel to alpha1. seven of which pack around the conserved central helix a3.  Vertebrate microtubule-associated protein RP/EB family member 3 (EBF3). The EB1 family is delineated by a highly conserved N-terminal domain with weak homology to the Calponin Homology domain. As a result. The sidechain forming the hydrophobic core of this bundle are highly conserved.  Vertebrate microtubule-associated protein RP/EB family member 2 (EB2 or RP1). The two alpha2 helices run antiparallel to helices alpha1 and form a similar fork in the opposite orientation and rotated by 90 degrees.  Vertebrate microtubule-associated protein RP/EB family member 1 (EB1).

Kakapo) would reveal that Shortstop is also a microtubule plus end tracking protein and that this activity is dependent of the presence of EB1. BLASTp searches of motifs similar to that found in APC revealed homology to the C-terminal domain of certain spectraplakin members. Binding studies would indicate that EB1 is capable of interacting with this BPAG1 motif in vitro. Our investigations of the EB1 – APC interaction delineated the respective binding regions as the conserved C-terminal EB1 domain and a series of two repeats localized to the APC C-terminus. As mentioned in the Cellular Functions of EB1. Subsequent studies by the Hammer lab have found this motif in melanophilin which is also recruited to microtubule tips by EB1. Prediction of this motif in CLASP members has also been noted.homology domains and EB1 microtubule plus end binding calponin homology domains are evident. Analysis of the single spectraplakin found in Drosophila. Shortstop (Shot. We began our investigations of factor recruitment focusing on the EB1 family. Thus. motor proteins. namely the human BPAG1 protein. Moving outwards from the direct interaction with the microtubule plus end. and signaling factors. the plus-tip members also recruit a host of cellular factors including polarity factors. That a similar face of a single domain would be used to interact with structurally unrelated cytoskeletal scaffolds is of great intrigue. kinetochore components. the C-terminal domain of EB1 serves to recruit this cell-polarity factor to microtubule plus ends. this includes the Rho exchange factor DRhoGEF2 but also the Adenomatous Polyposis Coli (APC) protein (of b-catenin destruction fame) and the molecular motor proteins NCD and Klp10a. This established the conserved region in APC and Shortstop as an EB1-binding motif. however one face of each domain is highly conserved although divergent between the two classes. .

Both structures reveal a novel dimerization domain fold characterized by a coiled-coil that folds back at its C-terminal end to form a fourhelix bundle region. structure of the EB1 conserved C-terminal domain was determined to high resolution in two different space groups.3 Structure of the EB1 conserved C-terminal domain . four-helix bundle junction characterized by a Phe-Tyr-Phe motif. Mutational analysis reveals that these residues are central to the EB1-binding motif interaction. The homodimerization of EB1 indicates that EB1 can likely bind more that one cargo simultaneously. Fig.Structure of the EB1 conserved C-terminal domain To reveal the determinants of EB1 cargo recruitment. The domain is two-fold symmetric down the axis of the coiled-coil and alignment of the two structures indicates plasticity within the fourhelix bundle region. The structure presents a highly conserved solvent-exposed hydrophobic region at the coiled-coil.

thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end (Slep. although diverse in structure. share a common property of multimerizing tubulin. While tubulin binding can be conferred by a single +TIP domain. and in vivo plus end tracking assays. In the case of EB1 and CLIP-170 members.It is evident that numerous proteins are recruited to the microtubule plus end and the mitotic spindle by these four plus end binding families. the promotion of microtubule nucleation and polymerization in vitro and plus end tracking in vivo require multiple domains acting in concert. Three main +TIP classes have been identified (XMAP215. The biochemical mechanisms of three plus end tracking protein families (EB1. 2007). Functional studies. Five crystal structures are presented of +TIP tubulin binding domains across multiple species and families illustrating structural conservation within families and functional convergence across families. are analyzed in vitro tubulin binding/assembly assays. in most cases. CLIP-170. EB1 and CLIP-170). but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically-induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and. two domains are . It is proposed that +TIPs. CLIP-170 and XMAP215) using x-ray crystallography. In vitro and in vivo analysis reveals an underlying general theme to these unrelated domains. plus end tracking in living cells. and EB1 Microtubule plus end binding proteins (+TIPS) localize to the dynamic plus ends of microtubules where they stimulate microtubule growth and recruit signaling molecules. Structural basis of microtubule plus end tracking by XMAP215.

a model is presented in which unrelated tubulin binding domains have convergently evolved a similar mechanism for enhancing tubulin assembly on growing microtubule ends by acting as multivalent tubulin polymerization chaperones (Slep.required for robust plus end localization and these two structurally unrelated domains can even cooperate if artificially joined together to achieve this activity. Mutagenesis suggests that several residues around one hemisphere contribute to tubulin binding. the CH domain of EB1 is spherical and approximately half the size. The tubulin binding domains of these three +TIP proteins are quite diverse in their architecture and show no evidence of a common evolutionary origin. highly conserved loops on one face of the TOG domain. the length of the TOG domain is fairly closely matched to a tubulin monomer (α or β) and likely makes extensive contacts through a combination of hydrophobic and electrostatic interactions. 2007). However. CLIP-170 also has a globular structure and the data suggests that it might bind tubulin’s disordered C-terminal tail as part of its binding interface (Slep. However. multimerization of TOG domains clearly facilitates tubulin nucleation in vitro and overall microtubule association in living cells. The different architectures also suggest varying interactions modes with tubulin and potential synergy between the +TIPs. From these data. XMAP215 has a very flat binding interface created by a series of rigid. This suggests that EB1 might nestle in the groove between tubulin protofilaments. as evidenced by the near superimposition of yeast Bim1p with human EB1 and yeast Stu2p with Drosophila Mini spindles. 2007). the structural conservation within a +TIP class is very high. XMAP215 members are more complicated. perhaps due to the relative weaker association between TOG domains and tubulin in higher eukaryotic members. In contrast to the elongated TOG domain. .

tubulin binding domains are arrayed in tandem along a polypeptide chain with presumably unstructured linkers in between (e. XMAP215 has a potent microtubule nucleation ability compared with CLIP-170. CLIP-170.g. For example. The cis linking domains within a polypeptide appears to be critical. EB1 family and Bik1 (the CLIP-170 homologue from yeast)]. Mini spindles. CLIP170). Native dimerization sequences are not essential. cis versus trans arrangements of +TIP domains may produce certain unique outcomes. the linear arrays of TOG domains in the extended XMAP215 structure may generate a pseudo protofilament-like arrangement that is particularly effective for microtubule nucleation. as a variety of artificial dimerization strategies (e.Comparison of the +TIPS and protein engineering studies suggest a considerable structural variation in how +TIP proteins can be interconnected to achieve their activities in plus end tracking and promoting tubulin assembly. Ch-TOG. and FKBPrapamycin-FRB) are capable of reconstituting +TIP protein function (Slep.g. 2007). However.g. which has tandem Cap-Gly domains and is dimerized via a coiled coil and Stu2p which has tandem TOG domains. In other cases. since it is shown that TOG domains added in trans do not reconstitute function. potentially related to an in vivo nucleation activity given XMAP215’s TACC-dependent localization to the centrosome. Analogous mechanisms exist for the actin cytoskeleton where the . In some cases. Studies reveal structural variation in how multiple +TIP tubulin binding domains can be combined to promote tubulin oligomerization for microtubule assembly.. Indeed. single α/β tubulin binding domains are found in a polypeptide and multivalent tubulin binding is achieved by polypeptide dimerization [e.g.. a dimerization domain and an additional C-terminal microtubule binding domain). gluthione S-transferase. Hybrid strategies are also employed (e. GCN4 leucine zippers.

promote microtubule growth in vitro and are requisite in vivo for microtubule plus end localization. This general idea was first introduced two decades ago by (Gard. This mechanism also enhances the spatial and temporal regulation of microtubule assembly in the cell. Studies show that single +TIP tubulin binding domains do not promote microtubule nucleation or growth in vitro (and in some cases are inhibitory). Multimerization overcomes the inherent polymerization barrier tubulin heterodimers face due to single longitudinal and lateral tubulin:tubulin affinities estimated to be on the order of mM and M respectively. such chaperones would become particularly important for enhancing the growth of microtubules when free tubulin dimers are below the critical concentration of microtubule assembly or when microtubule destabilizing proteins are active (e.g in mitosis). 2007). Physiologically. +TIP-induced multimerization of tubulin would increase the effective affinity for the microtubule lattice through cooperative binding. cylindrical lattice. 1987) to explain the high rates of tubulin assembly induced by XMAP215. By stabilizing tubulin-tubulin interactions prior to their full incorporation into a mature. the +TIPs would act as polymerization chaperones. In contrast. thereby decreasing the critical concentration for polymerization. For EB1 and CLIP-170. multimerized +TIP tubulin binding domains are potent microtubule nucleator in vitro. it was dynamically shown that plus end tracking requires an ability to bind more than one tubulin subunit.Spire protein utilizes several arrayed actin binding domains to template the nucleation of an actin filament (Slep. nor do they localize to microtubule plus ends in vivo. These results suggest a model in which +TIPs bind multiple tubulin dimers in solution and then deliver these larger tubulin oligomers to the ends of microtubules. in part by regulating the .

If folded into a HEAT-like structure. Tandem Cap-Gly domains in CLIP-170 have been shown to plus end track whereas single Cap-Gly domains of the homolog CLIP-115 show greatly reduced microtubule association. the conservation profile of alternating loops would be localized to one face of the domain. the basic unit of polymerization (Slep. Support for CLASP’s possible polymerization chaperone role comes from studies that observed CLASP-dependent microtubule subunit incorporation into fluxing kinetochore fibres (Slep. which contains only one TOG domain at its N-terminus. although poorly conserved at the primary structure level. 2007). +TIP families show divergent effects on microtubule dynamics. However. The model for +TIPs as multimeric tubulin chaperones undoubtedly oversimplify the complexity of interactions that are occurring at the microtubule plus end in vivo. some . suggesting that TOGL domains may bind tubulin by a mechanism similar to what is described in this study for the TOG domain. XMAP215 and CLASP appear to be ancient +TIP relatives that may employ a similar general strategy for plus end tracking. informed by TOG structures and secondary structure predictions several TOG-like (TOGL) domains can be identified in the CLASP family. 2007). a result they interpreted as XMAP215-facilitated incorporation of tubulin oligomers onto the microtubule end (Slep. Two additional dodeca-helical domains are predicted with alternating loops that exhibit high homology to TOG domain intra-HEAT loops. Optical trapping studies analyzing microtubule growth showed step-wise growth that increased in size in the presence of full length XMAP215. 2007). An apparent exception to the multiple tubulin binding rule for +TIPs is the CLASP family.localization and/or activity of +TIPs without modifying tubulin. Thus.

A major reason for this lack of knowledge is that prominent +TIPs. do not use well-defined. may lead to distinct effects on microtubule dynamics by preferentially stabilizing lateral versus longitudinal tubulin-tubulin interactions. and MCAK. As an example. and x-ray structures of +TIP-tubulin complexes will be required to further understand how +TIPs regulate the microtubule lattice (Slep. Multi-component in vitro assays. folded domains . a general molecular basis of microtubule tip tracking for the majority of EB1binding partners. CLASPs. An EB1-Binding Motif Acts as a Microtubule Tip Localization Signal The structure of the C-terminal domain of EB1 has been elucidated by X-ray crystallography (Honnappa et al.promoting growth while others act as anti-pause. arrayed versus dimerized. such as APC. Some of these differences in activity may reside in variations in the affinity constants of +TIP domains with tubulin monomers. The spatial arrangement of +TIP domains. the intricate web of +TIP-+TIP interactions may generate unique outcomes on tubulin assembly. Finally. 2007 and Dixit et al.. MACF. which do not contain CAP-Gly domains. Slep et al. STIM1.. destabilization. rescue and perhaps even nucleation factors. 2005.. 2005). non-EB1 family microtubule-binding CH domain proteins. including CLAMP and HEC1 appear to have higher affinity for the microtubule lattice than EB1 and localize along the entire length of the microtubule rather than just at plus ends. However. Weisbrich et al. and a structure-function relationship of its interactions with the cytoskeleton-associated protein-glycinerich (CAP-Gly) domains of CLIP170 and p150glued has been recently established (Honnappa et al. 2009). high resolution EM analysis of microtubules. 2006. oligomers and microtubules.. remains elusive. 2007)..

2005). To create the tightly focused. which makes both functional and structural studies challenging. Even though these two motors have overlapping functions.for EB1 targeting. in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. whether or not they define a general motif for +TIP interaction with EB1 and for localization to microtubule ends. because they are released from centrosomes or directly generated from chromosomes. whereas dynein has a dominant function in transporting K fibers to the centrosomes.. Studies reveal the roles of two minus end-directed motors.. Based on a crystal contact artifact seen in uncomplexed EB1c crystals and mutagenesis.. Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins During the formation of the metaphase spindle in animal somatic cells. kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes. dynein and Ncd. Honnappa et al. Work with APC and MACF suggested that these two proteins both contain an intrinsically unstructured sequence region of approximately 40 residues that binds to EB proteins in vitro (Bu and Su. Ncd is primarily responsible for focusing K fibers. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the . A novel localization of Ncd to the growing tips of C-MTs is reported. showed that an Ile-Pro dipeptide is important for the interaction of an APC-derived polypeptide fragment with EB1c in vitro (Honnappa et al. EB1. that is shown is mediated by the plus end-tracking protein. K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. 2003. However. diamond-shaped appearance of the bipolar spindle. 2005 and Slep et al. it was left open whether these sequence regions and amino acid determinants regulate the microtubule plus-end tracking activity of APC and MACF in cells and. 2005). more importantly.

Ncd also was recently observed to bind to an EB1 affinity column (Rogers. However. It was found that the nonmotor 'tail' domain (aa 1-290) of Ncd can bind directly to the COOH terminus domain of EB1 (EB1-C. albeit weakly. even the subset of growing microtubule never accumulated Ncd-GFP-NES at their tips (Goshima. However. After EB1 depletion by RNAi in Ncd-GFP-NES cell line. no plus end accumulation of Ncd-GFP-NES was seen. suggesting that the motor domain may augment affinity for the microtubules and thereby aid plus end localization (Goshima. Next. suggesting that the Ncd tail and APC bind to the same site on EB1.. From these results and simulations. 2005). APC adenomatous polyposis coli protein). instead the microtubules were evenly labeled with this protein. No specific mutagenesis strategy was availabe for eliminating plus end tracking of Ncd while retaining its other critical mitotic activities such as microtubule cross- . EB1 is a highly conserved microtubule plus end-tracking protein that binds various cargo proteins (e. a model is proposed on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis (Goshima. 2005). 2004). This binding was competed by addition of a fragment of human APC protein (2744-2843 aa) that binds to EB1's COOH-terminal domain. 2005). expression of the Ncd tail domain (1-290 aa) fused to GFP did not track along microtubule plus ends in vivo. aa 208-278). microtubules become less dynamic and frequently enter a pause state where they exhibit minimal growth or shrinkage.g. After EB1 RNAi. It was therefore of interest investigated whether the plus end accumulation of Ncd-GFP is mediated by EB1.capture and transport of K fibers along C-MTs. an in vitro interaction between purified Ncd and EB1 was tested using a GST pull-down assay.

the consequences were tested of pole focusing and Ncd-GFP localization in mitosis after RNAi of EB1. 2005). it is proposed that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking. When these phenotypes were examined quantitatively. and (3) K fiber transport on C-MTs. Based on live cell imaging and computer simulation analyses. RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus enddirected motor proteins in these processes. indicating that K fiber binding does not require EB1. it was found that the EB1 phenotype consists of pronounced K fiber defocusing and has less pronounced centrosome detachment. Time-lapse imaging of mitotic EB1 RNAi cells showed no plus end microtubule enrichment. Qualitative study of EB1 RNAi found both centrosome detachment and pole defocusing. Although not definitive proof because EB1 RNAi causes microtubule dynamics defects in addition to displacing Ncd from the microtubule plus end. . By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type. In this setting of EB1 RNAi where Ncd was mislocalized from microtubule plus ends but not the spindle.bridging. centrosome detachment and K fiber focusing was examined. 2005). (2) 'search and capture' of K fibers by the tip of growing C-MTs. However. However. as expected from the observed interphase results. it was found that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing (Goshima. this result is consistent with a functional link between EB1-dependent localization of Ncd to microtubule tips and K fiber coalescence (Goshima. which is more similar to the Ncd RNAi than the dynein RNAi phenotype. Ncd-GFP still strongly and dynamically (revealed by FRAP) localized to spindle MTs.

Albeit less efficient than dynein. Ncd's major role is likely to be in inter-K fiber crosslinking.RNAi results suggest that Ncd plays a role in K fiber focusing through three mechanisms described above. 2005). Ncd also likely contributes to minus end-directed transport of K fibers along C-MTs. which show that these motors are associating and dissociating with K fibers on a rapid time scale and thus are not behaving as static cross-linkers. Finally. as evidenced by finding that Dhc64C RNAi causes detachment of centrosomes from the minus ends of K fibers. as evidenced by the splaying of K fibers after Ncd RNAi and the prominent localization of Ncd-GFP to K fibers. It is believed that this K fiber focusing effect also primarily involves dynein's role as a transporter of K fiber bundles . dynein also contributes to the focusing of the minus ends of K fibers. because K fibers are continually splaying and coalescing. It is also showm that the process of lateral K fiber interactions is highly dynamic. Although secondary to Ncd. This process occurs in the absence of centrosomes and C-MTs. Such observations are also consistent with FRAP measurements of Ncd-GFP. Cytoplasmic dynein in S2 cells plays a dominant role in transporting K fibers along microtubules. it is believed that Ncd at the tips of C-MTs may act to capture K fibers and facilitate subsequent minus end transport of the K fiber (Goshima. since Ncd/Dhc64c double RNAi causes very severe splaying of K fibers. because acentrosomal spindles created by centrosomin RNAi also show severe K fiber unfocusing when Ncd is also knocked down by RNAi. This role of dynein becomes particularly clear after Ncd depletion. because the centrosome to K fiber distance is somewhat greater in the Ncd/Dhc64C double RNAi compared with Dhc64C alone. This process most likely involves cross-linking of microtubules by Ncd's force generating motor domain and its positively charged 'tail' domain that also binds to microtubules independently of the motor.

g. its ability to bind two microtubules and its slow motor activity makes it an effective cross-bridger between microtubules in the spindle. small numbers of dyneins could rapidly transport K fibers along C-MTs. would still enable dynein to effectively transport the K fibers along the C-MTs (Goshima. as shown by FRAP data. processive motor. However. Instead. Consistent with this idea. The molecular properties of kinesin-14/Ncd and cytoplasmic dynein are well designed to support the above proposed functions of these two motors in pole focusing. such as potentially transporting and concentrating cross-linking proteins at minus ends of K fibers. Phenotypic RNAi analyses may account for differences in the pole unfocusing phenotypes of Ncd or dynein depletions that have been described in the literature. slow motor that is not designed for cargo transport. the rapid on-off rates of these cross-bridges. as well as for crossbridging of C-MTs plus ends to K fiber. Thus. These properties are likely to be advantageous for inter-K fiber cross-linking. is particularly important for K fiber focusing. may become more crucial when centrosomes are detached or absent from the spindle. the most dramatic pole unfocusing phenotypes for kinesin-14 mutations/depletions have been described in plant mitosis and animal . Ncd is nonprocessive. the presence or absence of centrosome and differences in microtubule dynamics) may determine whether Ncd or dynein acts as the essential contributor to pole coalescence. Ncd/kinesin-14 function. 2005). which causes the coalescence of most peripheral K fibers toward the centrosome as shown in computer simulations.. spindle architecture in the given system (e. synthetic effects of kinesin-14 and dynein motors on pole focusing have been reported in centrosomefree spindles reconstituted inXenopus egg extract (Goshima. 2005).along C-MTs. For example. dynein may have other roles in K fiber coalescence. Indeed. Specifically. However. In contrast. Cytoplasmic dynein is a fast.

. if C-MTs are very abundant. Even though it was found that C-MTs are still dynamic after Ncd RNAi.' facilitates their connection to K fibers. which is not observed for Ncd. as shown convincingly in Xenopus extract system. However. 2005). 2005). The enhanced K fiber unfocusing in EB1 RNAi-treated cells. the high probability of close approximation of K.g. Microtubule dynamics. The microtubule plus end tracking of Ncd. It is proposed that plus end tracking of Ncd on newly nucleated C-MTs. suggests that plus end tracking of Ncd may serve a function in pole focusing. specifically the relative number of K. The accumulation of kinesin-14 at the plus end overlap zone in mitotic spindle has been shown but whether this localization reflects localization of plus ends of individual microtubules is not known. In contrast. it is possible that Ncd at the plus end also modulates microtubule C-MTs in the bipolar spindle. which displaces Ncd from plus ends but not K fibers. Localization of a kinesin-14 motor protein to the plus end of interphase microtubules has been recently reported in plants. as a 'capture factor. dynein also is likely to play crucial roles in pole coalescence in some acentrosomal spindles.. also may alter the relative contribution of the two motors. and kinesin-14 is less important for pole focusing in such cells (e. For example. but it is more enriched on the depolymerizing microtubules.meiosis. Yeast Kar3p also was shown to accumulate at the plus ends of microtubules at the shmoo. systems in which spindle assembly occurs through a centrosomeindependent mechanism and in which interactions between C-MTs and K fibers are simply absent. Ncd is nonessential in fly development).and C-MTs may enable dynein to easily link these two networks without any assistance from kinesin-14 motors at microtubule plus ends (Goshima. somatic animal cell mitosis utilizes centrosomes. as does EB1 or other tiplocalized proteins (Goshima.

It is also noted that the simulations are two-dimensional and encounters between C-MTs and K fibers would become less likely in three dimensions. 2005). Ncd at the tips of growing microtubules may act to capture microtubules that arise from the opposite pole. The microtubule plus end search-and-capture mechanism might apply to other aspects of metazoan cell division. This idea is analogous to a 'search and capture' model for how C-MTs find chromosomes. Ncd may generate a connection between K fiber and C-MTs temporary. and thereby facilitate the recruitment of minus end-directed transporter (primarily dynein but Ncd contributing as well) for the transport of K fibers. Additionally. In this case. and genetic study demonstrates that Ncd produces an inward force on antiparallel microtubules during early mitosis. and one might expect the effect of a C-MT-mediated capture/transport mechanism to become more important under such circumstances (Goshima.possibly using its second microtubule binding site located in its NH 2-terminal tail domain. the tip-localized motor Ncd enables C-MTs to 'search' for and then 'capture' a second major microtubule network in the spindle. 2005). the K fibers. Ncd at the plus end may act as a K fiber transporter once it binds. Another possible target of tiplocalized Ncd may be free microtubules. For example. cross-linking interactions between antiparallel microtubules occurs at overlap zone of microtubules. although this transport would be less efficient than that produced by fast and processive dynein motors. which are either released from centrosomes or generated de novo in cytoplasm and are eventually incorporated into the spindle by a dynein-dependent transport process (Goshima. An actin-microtubule cross-linker (spectraplakin) that contributes to organization of the microtubule network .

The results provide the first description of the dynamics of these important proteins and provide key insight about how they function during cytoskeletal cross-talk (Applewhite. but not with the plus end. however. In the cell periphery. little is known about the dynamics of spectraplakins and how they bridge actin filaments and microtubules. association with the microtubule lattice mediated by the GAS2 domain of Shot. Shot was found to interact with microtubules using two different mechanisms. Shot binds growing plus ends through an interaction with EB1. Spectraplakins are an ancient family of giant cytoskeletal proteins that are essential for a diverse set of cellular functions. In the cell interior. This cross-linking maintains microtubule organization by resisting forces that produce lateral microtubule movements in the cytoplasm. Shot is able to interact with microtubules via two distinct mechanisms. and this pool of Shot is actively engaged as a cross-linker via its NH (2)-terminal actinbinding calponin homology domains.The dynamics of actin and microtubules are coordinated in a variety of cellular and morphogenetic processes. yet. through multiple EB1interacting motifs at the COOH terminus of Shot. The interaction of Shot with the microtubule lattice. requires an intact network of F-actin and a functional actin-binding domain at its NH2 terminus. 2010). Shot crosslinking couples microtubules to actin in order to resist motor-mediated lateral sliding movements. The data suggest that Shot can exist in at least two different . little is known about the molecules mediating this cytoskeletal cross-talk.and microtubule-binding domains cooperate to regulate microtubule dynamics by cross-linking the two cytoskeletal networks. and an interaction with growing plus ends that is mediated by EB1. This study describes the intracellular dynamics of Shot and a structure-function analysis of its role as a cytoskeletal cross-linker. The data also demonstrate that the actin. Shot associates with the microtubule lattice via its GAS2 domain.

Understanding how Shot is regulated will be an important next step. the molecule opens and is able to bind the lattice and actin. . so that the timing and placement of Shot-mediated crosslinking is precisely controlled within the cell. This result suggests that GAS2 exerts a dominant effect when present in the same molecule and further suggests that it must be actively repressed when Shot is at microtubule plus ends. In the second state. binding of the CH domains to actin may itself be sufficient to trigger the transition from tip-tracker to lattice-binder. perhaps activation of this pathway leads to activation of the cross-linking activity. 2010). Second. It is speculated that the interconversion between these two states represents a regulated conformational change. observed after actin depolymerization or with the C isoform that exhibits low affinity for actin. or Shot-ΔRod isoforms in the actin-rich region of the peripheral lamella. In the first state. Fourth. there is evidence that Shot may act downstream of the small GTPase Rho. First.states. Shot is bound to the microtubule lattice via the GAS2 domain. Shot preferentially interacts with microtubule plus ends. Third. Understanding when and where Shot is activated to cross-link will be key to understanding its cellular functions (Applewhite. The hypothesis is favored that Shot is regulated by an intramolecular interaction that simultaneously represses the GAS2 and actin-binding CH domains during tip tracking. suggesting that calcium may participate in regulation of microtubule lattice binding. B. observed with the A. but several potential mechanisms immediately present themselves. it is interesting to note that the Shot-GAS2-CTD construct decorated the microtubule lattice despite the presence of the EB1interaction motif. the EF-hand motifs are closely positioned to the GAS2 domain. The results suggest that the pool of Shot that is actively serving as a cross-linker is the lattice-bound population. On activation.

2008). especially if the motors associated with the surface of one organelle engage multiple microtubules. Shot is. Previous studies of the role of mouse ACF7 have clearly demonstrated a role for this protein as a 'guide' to direct microtubule growth along actin stress fibers to assist them in targeting to focal adhesions (Wu. Different modes of microtubule association may also allow spectraplakins to regulate microtubule dynamics in different ways.Shot possess these dual mechanisms (tip vs. by cross-linking microtubules to the actin network to prevent lateral microtubule displacement by motor proteins (Applewhite. Microtubule dynamic instability is used as a mechanism to search intracellular space for docking and stabilization during kinetochore microtubule capture and polarization of migrating cells. in S2 cells. instead organizing acentriolar networks to organize their cytoplasm. These forces may be generated during organelle transport. therefore. +TIPs are perfectly positioned to influence the conformation of tubulin at this site. acentriolar cells rely upon actin-microtubule cross-linking to maintain their organization and to resist forces produced by motor proteins. A common theme emerging from studies of other +TIPs is that many of them function is as regulators of microtubule dynamic instability. in the absence of a structural anchor at the minus end. Perhaps association with the microtubule plus end allows spectraplakins to search for activating inputs at the cell cortex or in actinrich subregions of the cell. It is speculated that. Shot-mediated cross-linking performs an additional function by crosslinking microtubules to actin in order to resist whip-like lateral displacements by motor proteins. This study presents evidence that Shot also can influence microtubule dynamics through a different mechanism. Many Drosophila cell types exist without a dominant interphase microtubule organizing center. 2010). Because the majority of subunit addition and loss occurs at the plus end. lattice) for microtubule interaction. The current data show that. .

likely to play an important role in maintaining microtubule organization for the efficient motor-mediated delivery of organelles and other cargo to specific destinations within the cell. 2010). releases from EB1. and engages actin with is dual CH domains and the microtubule lattice with its GAS2 domain. Interaction with EB1 (End binding protein) is required for axonal growth-promoting roles of Shot at polymerizing plus ends of microtubules. Key regulators of microtubules in this context are the spectraplakins. Testing these ideas will require careful observation of Shot dynamics in the developing embryo where cells are responding to extracellular cues. On receiving the activating stimulus. Furthermore. Based on the current data a speculative model has been developed in which 'inactive' Shot associates with a growing microtubule plus end by its interaction with EB1. In this conformation. Dynamic instability allows microtubules to probe the cytoplasm for an activating signal in a 'search-and-capture' mechanism. this activity is consistent with the observation that the microtubule network in mushroom body neurons in Shot mutants exhibits gross disorganization and an overall loss of microtubule polarity. Microtubule-mediated axonal growth and EB1 The correct outgrowth of axons is essential for the development and regeneration of nervous systems. a family of evolutionarily conserved actin-microtubule linkers. as well as genetic analyses to determine how the EB1-Shot interaction contributes to development (Applewhite. it is able to cross-link microtubules to actin to influence their growth trajectory or to stabilize the network against forces produced by motors. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Shot changes its conformation. Axon growth is primarily driven by microtubules. .

This function is EB1-independent but requires net positive charges within C tail which essentially contribute to the microtubule shaft association of Shot. firmly establishing Shot-EB1 interaction as an important determinant of . MT polymerization. it is reported that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. plus end regulators) and structural MAPs (microtubule-associated proteins). MT-based transport and MT-actin cross talk have been highlighted. and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. 2012).Binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail). spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins. Therefore. loss of EB1 function phenocopies Shot loss. In addition. Many MT-binding proteins have been identified and their potential roles in regulating the key processes of MT stabilization. Second. Shot regulates MT polymerization dynamics and guides them in the direction of axonal growth. ranging from axonal growth to neurodegeneration. Shot stabilizes MTs. But how these different functions merge into coordinated MT network regulation is poorly understood (Alves-Silva. a role generally assigned to structural MAPs. From these data a model is deduced that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth (Alves-Silva. The regulation of MT networks is essential for many neuronal functions and processes. However. In this function. understanding how MTs are regulated in neurons remains a major challenge. 2012). Spectraplakins are key integrators of different MT regulatory processes. First. such as Tau. Shot interacts with EB1. mutations of these motifs abolish Shot functions in axonal growth. MAP2 or MAP1b.

This finding might have important implications. studies in . studies reveals the importance of the C tail for Spectraplakin function. and this could be caused by functional compensation through spectraplakins.microtubule guidance as a crucial mechanism underpinning axon growth. For example. Shot acts as an actin-MT linker during axonal growth. In addition. 2012). strongly suggesting that work on spectraplakins will provide exciting new opportunities to unravel the complexity of MT regulation in neurons (Alves-Silva. 2012). Shot. as deduced from its crucial roles both in MT stabilization and guidance and its requirements in both neurons and tendon cells (Alves-Silva. Shot localization along the shafts of axonal MTs is important for MT-stabilization. At the molecular level. It remains to be seen whether such potential functional overlap is general or contextspecific. loss of structural MAP functions has relatively mild phenotypes even in double knock-out mice. MT-stabilizing roles in neurons have similarly been reported for the Shot homolog BPAG1. Thus. adding a third crucial MT regulatory role to the list. MT-stabilizing roles of spectraplakins appear conserved in mammals. Therefore. While GRD had previously been established as the domain of Shot which has the widest functional requirements. different MAPs and spectraplakins might display individual traits. axon shortening caused by knock-down of ACF7 in primary cortical neurons or N2A cells was rescued by taxol application. such as dependence on different MT modifications or selective antagonism to different destabilizing factors (Alves-Silva. Ctail is similarly important. +TIPs and actin-binding proteins. The domain requirements underlying MT stabilization are conserved. suggesting that spectraplakins might functionally overlap with structural MAPs. spectraplakins work at the crossroads of structural MAPs. For example. Furthermore. 2012). Thus.

GSK3 (as a regulator of APC downstream of Wnt3a/Dvl1 and of CLASP). Such a mechanism is consistent with other models for MT association and can explain why C tails are not conserved at the amino acid sequence level. In support of this model. In the absence of ShotEB1 complex function. Similar phenotypes of curled. 2012). and most contain MtLS motifs. MTs are disorganized and axons extend shorter. Despite functional conservation. the C terminus of mouse ACF7 has recently been demonstrated to detach from MTs. The favored explanation for why MT disorganization attenuates axon growth is that MTs are less efficient in pushing . Studies have identified a role for the arginines of Ctail and proposes that this positive net charge attracts C tail to negatively charged MTs. They are all unlikely to form an ordered secondary or tertiary structure. when negative surface charges of MTs were enzymatically removed (Alves-Silva. they all display a high content of arginines. The second subcellular role of Shot in MT guidance establishes EB1 and the EB1Shot complex as important determinants of axonal growth. serines and glycines. Therefore. such as in growing axons and tendon cells (Alves-Silva. and in each case the C tails enhance their MT association. and this work has provided first experimental proof that it is functionally relevant in vivo. C tails are not conserved at the protein sequence level but display other commonalities instead. this mechanistic principle appears conserved across a range of homologous proteins. criss-crossed MTs correlating with axon shortening have been described in mammalian neurons lacking ACF7 function but also defective for Dynein/Lis1.fibroblasts have shown that related C termini of the mammalian spectraplakin ACF7/MACF1 and of human Gas2-like1/hGAR22 and Gas2-like2/hGAR17 all display MT-stabilizing properties that are dependent on their GRDs. 2012). or Spinophilin/Doublecortin.

and this interaction is likewise believed to be required to steer MT polymerization events in elongating axons (Alves-Silva. These findings do not only advance the principal understanding of cytoskeletal regulation during axonal growth. 2012). the F-actin-binding factor drebrin was shown to interact with EB3. Recent studies reveal that +TIPs compete among each other for interaction with EB1 at MT plus ends. For example. such as the actin cortex in the axons or actin networks or bundles in growth cones. the model for MT guidance displays interesting commonalities with models for ACF7 function in migrating keratinocytes during wound healing.along the axonal axis in the direction of axon growth. It is therefore proposed that Shot performs its MT guiding roles by linking MT plus ends to actin structures. skin blistering and cell migration in wound healing and brain development. spectraplakins play roles in clinically relevant cellular processes including neurodegeneration. Therefore. APC. but can be extrapolated to other functions of spectraplakins. such as CLASP. Shot works at the cross-roads of different mechanisms of MT regulation have unraveled the underpinning mechanisms using a genetically and experimentally amenable and functionally well conserved Drosophila model of axon growth. For example. where ACF7 is suggested to guide . Other +TIPs. Such a function of spectraplakins is likely to coexist with parallel mechanisms of actin-MT linkage. Dynein/Lis and CLIPs. are present in neurons and contribute to axonal growth regulation. The two modes of Shot function proposed in this study may very well be applicable. Thus. providing new opportunities to gain an understanding of regulatory +TIP networks in the context of axonal growth (Alves-Silva. 2012). Studies show roles of Shot in MT guidance and axon growth also essentially require its linkage to actin. the +TIP functions of spectraplakins can now be analyzed in the context of other +TIPs.

Notably.MTs along actin stress fibers to focal adhesions. the subcellular mechanisms of spectraplakins not only have implications for the understanding of axonal growth but also for their other functions in neurons and non-neuronal cells (Alves-Silva. whereas MtLS motifs and actin-binding calponin-homology domains of Shot are dispensable. are required for uniform dendrite microtubule polarity. where they enhance each other’s localization. it was shown that kinesin-2. For example. GRD and Ctail are essential in tendon cells. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity. Furthermore. microtubules are organized into polarized noncentrosomal arrays. Using RNAi and genetic approaches. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites. It was found that growing microtubules navigate dendrite branch points by turning the same way. which are often highly branched. MT-stabilizing roles are likely to explain Shot function in Drosophila tendon cells. mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. this system was used to understand how cells can maintain dynamic arrays of polarized microtubules. Therefore. toward the cell body. yet few mechanisms that control these arrays have been identified. tendon cells have been proposed as a cellular model for support cells of the vertebrate inner ear that are known to express the Shot homolog BPAG1. the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to . Moreover. Just like MTstabilizing roles of Shot in axons and fibroblasts. Role of directed microtubule growth and +TIPs in uniform microtubule polarity in dendrites In many differentiated cells. 2012). and the +TIPS EB1 and APC.

study demonstrates that growing microtubule plus ends almost always turn toward the cell body at branch points and that they track stable microtubules through branches. On the basis of these results. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie. therefore it is proposed that a growing microtubule plus end coated with EB1 is transiently linked. through the interaction between Apc and the EB1 tail. It is proposed that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. 2010). 2010). Kinesin-2. SxIP motifs in Apc and Klp68D could also contribute to this interaction. and APC are all required for maintaining microtubule polarity and are linked in an interaction network (Mattie. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2. the motor domain would be free to walk along a nearby stable microtubule toward the plus end and cell body (Mattie. to kinesin-2 as it passes through the branch. . EB1.branch points suggests that these proteins work together at dendrite branches. Apc can interact with the Kap3 subunit of kinesin-2 and so could increase concentration of the motor near the branch. APC. a model for directed growth of microtubules in dendrites is proposed. EB1-GFP does not concentrate at branches. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Using Drosophila dendrites as a model system. and EB1 are likely to play a role in this process. 2010). The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. Because both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail.

Observations of plus-end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved. This mechanism is also probably necessary to establish uniform microtubule polarity in branched dendrites. Once the tip of the microtubule turns. so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Studies reveal that kinesin-2 has shorter run lengths than kinesin-1 and that individual EB1 interactions with the microtubule plus end persist for less than a second (Mattie. For example. Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. they probably represent a switch from a freely growing plus end to one that is following a track (Mattie. Alternatively. a kinesin anchored to the cell cortex by its C terminus could shuttle minus-end-out microtubule seeds into dendrites. and a similar mechanism could play a role in dendrites. Stable microtubules do not accommodate such sharp turns. 2010). but it probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity. 2010). polarized microtubule nucleation sites could be localized within . growth should be constrained by the dendrite walls.Even a very brief application of force pulling the growing microtubule toward the cell body should be sufficient to steer growth toward the cell body. Instead. frequently plus ends are seen turning sharply. The association of the growing plus end with a stable microtubule would probably only need to be maintained over a distance of a micron.

and it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. and polarized microtubules are found in many cell types. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells. the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be involved in aligning microtubules in many circumstances (Mattie. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. Thus. This is a newly identified function for both the +TIP proteins and kinesin-2. directed growth of microtubules could also be important during axon outgrowth. 2010). APC. A functional link between dynamic microtubules and endocytosis Many cell types including developing oocytes. In fact. and sliding of microtubule tips along the side of microtubules was also seen in this system. directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity.dendrites. 2010). The polarity of the oocyte is established by the specific localization of three . but there was no association with a particular direction of movement or overall microtubule polarity (Mattie. fibroblasts. Indeed. epithelia and neurons use mRNA localization as a means to establish polarity. Because kinesin-2. EB1. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie. this type of microtubule tracking behavior has been observed in axonal growth cones under low-actin conditions. 2010). Kinesin-2 has previously been shown to be enriched in the tips of growing axons in cultured mammalian neurons.

Oskar protein is required for recruiting EB1 and CLIP-190 to the posterior pole. the precise organization of the oocyte microtubule cytoskeleton remains an open question. EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte. In order to examine the polarity of oocyte microtubules. Oskar protein has been shown to induce the formation of long F-actin fibers at the posterior of the oocyte.critical mRNAs . Results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. One possibility is via the activity of cross-linking proteins . Thus. with additional foci detected within the oocyte cytoplasm and along the cortex. The mechanism by which Oskar performs this function is less clear. 2012). the localization of canonical microtubule plus end binding proteins. Surprisingly. Consistent with this hypothesis. Actin function in recruiting EB1 and CLIP-190 to the posterior pole. However. and the activity of microtubule motors. This suggests that EB1 and CLIP-190 recruitment to the posterior pole occurs downstream of Oskar protein localization and requires integrity of the actin cytoskeleton. Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. However. however. The localization of these mRNAs requires microtubule integrity.oskar. multiple polarity-determining pathways are functionally linked in the Drosophila oocytes (Sanghavi. bicoid and gurken. treatment of ovaries with Lat-A resulted in loss of EB1 and CLIP-190 foci from the posterior pole. Although posterior EB1 and CLIP-190 are not essential for oskar mRNA localization. It was therefore hypothesized that these actin fibers might recruit growing microtubule plus ends. Oskar protein was still detected at the posterior. it was found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. was visualized. Under these treatment conditions however.

capu and spire.that have been shown to bind microtubule plus ends as well actin. . Another possibility is that the actin network might be required for polarizing oocyte microtubules. but it nonetheless suggests that mis-expression of Oskar affects the dynamics of microtubules within the oocyte (Sanghavi. the ectopic Oskar focus contained high levels of Khc. Khc. Khc and Kin:βgal might label a population of microtubules that are polarized.Consistent with this notion. 2012). The reason for this phenotype is unclear. Ectopic expression of Oskar protein along the entire oocyte cortex or within a central focus failed to efficiently recruit EB1 and CLIP-190. was localized to the posterior in oskar protein-null mutants. Posterior localization of CLIP-190 was also affected under these conditions. The mechanism by which Oskar performs this function. Studies also suggest that Oskar is not sufficient for recruiting EB1 and CLIP-190. but EB1 and CLIP-190 were not. It is therefore likely that multiple factors function to recruit these plus end binding proteins to the posterior pole. yet relatively static in vivo. At present it is unclear whether Khc and Kin:βgal localization is truly indicative of microtubule plus ends. 2012). but to a lesser degree. Studies demonstrate that endocytosis within the oocyte is asymmetric. 2012). however. is largely unknown (Sanghavi. mis-expression of Oskar reduced the posterior accumulation of EB1. Similarly. but not EB1 and CLIP-190. occurring at a higher level at the posterior. Khc was found in the center of par-1 mutant oocytes. Oskar protein is required for maintaining this polarized distribution of endocytosis. In contrast to EB1 and CLIP-190. As mentioned previously. however. result in oocytes that lack microtubule polarity (Sanghavi. In addition to the ectopic Oskar focus. Curiously. mutations in the actin nucleators.

it is hypothesized that Oskar promotes endocytosis by recruiting microtubule plus end binding proteins to the posterior pole. as well as endocytosis was drastically reduced at the oocyte posterior in oskar protein-null and staufen mutant. These findings highlight the importance of microtubule plus ends and CLIP-170 in vesicle transport (Sanghavi. EB1 and CLIP-190 might deliver factors to the posterior that facilitate high endocytic activity (Sanghavi. the vertebrate homolog of CLIP-190. Based on these observations. CLIP-170 was also shown to function at microtubule plus ends to stimulate phagocytosis in macrophage cells and to promote the capture and minus end transport of melanophores in Xenopus). 2012).Growing microtubule plus ends are recruited to the oocyte posterior by Oskar. These results suggest an intimate relationship between dynamic microtubules and efficient endocytosis at the posterior pole. EB1 and CLIP-190 localization. was initially identified as a protein that directly bound to endocytic vesicles as well as microtubule plus ends. More recently. with an enrichment of plus ends at the posterior pole. it is interesting to note that CLIP-170. One possibility is that the dynamic microtubules themselves are important for posterior endocytosis. Treatment of egg chambers with a microtubule-stabilizing drug also had a similar effect on microtubule plus end localization and endocytosis. Consistent with this hypothesis. These results indicate that oocyte microtubules are polarized. The results also suggest that the primarily function of the polarized microtubule network is to promote high endocytic activity at the oocyte posterior (Sanghavi. 2012). Effects of Mutation or Depletion RNAi and antibody depletion of EB1 . In this regard. 2012). Alternatively.

on average. The rates of microtubule growth and shrinkage were 3. 2002).6 ± 3.8 ± 0. and 17. or pause to shrinkage) was 0.8 µm/min. with velocities of growth and shrinkage intermediate to those measured in mammalian cells and yeast (Tirnauer.9 µm/min and 8. Microtubule behavior was very different in cells depleted of EB1 by dsRNA. 1999).1 µm/min) . 2002).029 transitions from shrinkage (or pause) to growth per second. EB1 dsRNA-treated cells attached and spread as well as control cells and displayed no obvious morphological abnormalities.021 transitions per second. Rates of microtubule growth (3.7 ± 1.2% of the time in growth.5% of the time in shrinkage. These parameters of dynamic instability measured for S2 cell microtubules are similar to those measured in other cell types using GFP-tagged tubulin.0 µm/min) and shrinkage (8. These results represent the first direct measurements of microtubule dynamic instability in Drosophila cells (Rogers. The populations of microtubules in control cells spent.7 ± 2. To probe the effects of EB1 depletion more carefully. Microtubules in untreated control cells exhibited dynamic instability. 55.To gain insight into the cellular functions of EB1. whereas the rate of rescue was 0. live cell fluorescence microscopy was used to observe microtubule behavior in control and EB1 dsRNA-treated S2 cells transfected with GFP-tubulin. asynchronously transiting between phases of elongation and shrinkage. respectively. whether RNAi depletion of EB1 affected microtubule organization was examined by fluorescence microscopy. 27. When plated on concanavalin A-coated coverslips. Tubulin staining revealed that the interphase microtubule organization in these cells was indistinguishable from controls (Rogers. The rate of catastrophe (the switch from growth. Six days of dsRNA treatment was sufficient to reduce EB1 protein to very low levels.3% of the time in a paused state (neither growing nor shrinking).

were similar compared with untreated control cells. However, the frequencies of
catastrophe in EB1-depleted cells were approximately three fold lower compared
with control cells. The most notable effect of EB1 depletion was that microtubules
spent the majority (55.2%) of their lifetimes in a paused state relative to growth
(30%) or shrinkage (9.3%). These results indicate that EB1 promotes microtubule
dynamics in Drosophila cells. The effects of EB1 RNAi on microtubule dynamics
in S2 cells are qualitatively similar to interphase microtubule behavior observed
in bim1Delta S. cerevisiae as reported by Tirnauer (1999). In both cases,
microtubule catastrophe and rescue frequencies were decreased in the absence of
EB1/Bim1p and microtubules spent the majority of their lifetimes in a state of
pause (Rogers, 2002).
Given the role of EB1 family members in mitosis in yeast, how RNAi inhibition
of EB1 expression affected mitosis was examined in Drosophila cells. Mitosis in
untreated or GFP dsRNA-treated cells progressed in a very reproducible manner.
At prophase, the two spindle poles were in close proximity to condensing
chromosomes and always nucleated asters of long, radial microtubules. As the
cells proceed to prometaphase, the spindles assumed a typical bipolar organization
and chromosomes are positioned between each pole. At this stage, and for all
successive stages, bipolar spindles nucleate highly developed radial arrays of
astral microtubules, many of which extended to the cell cortex. The chromosomes
congressed to the metaphase plate, and subsequently migrated to the spindle poles
during anaphase and telophase, the two incipient cells assumed a more rounded
shape (Rogers, 2002).
In cells lacking EB1, defects in microtubule organization are readily apparent.
During preprophase, EB1-deficient cells duplicate centrosomes normally and the
two centrosomes migrated to opposite sides of the nucleus as in control cells. At

this stage, each centrosome nucleated a normal radial array of long microtubules
that extended toward the cell periphery. However, when dsRNA-treated cells
progress to prophase, the long cytoplasmic microtubules disappear, and instead,
only very short (<1 µm) astral microtubules are observed clustered around the two
poles. Short microtubule fragments unattached to the poles are often present in the
cytoplasm of EB1-deficient cells. These phenotypes are observed in 74% of the
EB1-deficient prophase cells examined, but were never observed in untreated or
GFP RNAi control cells. From these observations, it is concluded that EB1 is
required for stabilizing microtubules and creating astral arrays in mitosis (Rogers,
The loss of EB1 also produced aberrant spindle phenotypes in metaphase cells that
could be classified into four general categories. The most common defect was a
complete loss of astral microtubules. These spindles maintain their bipolar
symmetry, but commonly exhibit detachment of centrosomes from the spindle.
The second class of defects (observed in 33% of the cells) lacked astral
microtubules and exhibited an overall compaction of the spindle into a basket-like
meshwork of microtubules surrounding the chromosomes. In these structures, the
poles could not be clearly distinguished, but mitotic chromosomes maintained
their position at the center of the spindle. The third type of defect was a
detachment of a spindle pole from the bundles of microtubules that were
connected to the kinetochores. These spindles exhibited a 'splayed' morphology.
The fourth category of defect was 'barrel-shaped' spindles that maintained their
symmetry, but failed to focus the microtubules at the poles and also lacked astral
microtubules. These phenotypes did not appear to be due to gross centrosome
defects, as immunofluorescent staining with antibodies against centrosomin
protein revealed spindle poles to be present and intact. In all four classes of

defective spindles, the distance from pole to pole was significantly smaller (5.4 ±
1.1 µm) than in GFP dsRNA-treated cells (7.7 ± 0.9 µm). These results indicate
that EB1 plays a critical role during spindle assembly (Rogers, 2002).
The mitotic defects observed in the EB1 RNAi-treated cells were severe enough
that it was thought that they might affect cell cycle progression by activating the
spindle checkpoint. To test this possibility, fixed cells were stained for DNA and
the number of cells with mitotic figures was scored as a percentage of the entire
cell population. In EB1 dsRNA-treated cultures, the mitotic index was 5.9%,
approximately double that of control cultures at 2.7%. Although significant, this
difference was not as dramatic as might be expected if mitotic progression were
completely blocked. If the mitotic checkpoint were activated for prolonged
periods of time, an increase in apoptotic cells might be expected. However, S2
cells exhibit macrophage-like properties, and it was observed that they consume
their apoptotic neighbors, as judged by nuclear morphology. This property of S2
cells could give rise to artificially low mitotic index measurements. To determine
at which stage of the cell cycle mitotic progression is interrupted, all of the mitotic
cells in these samples were categorized according to their stage of mitosis. In
control-treated cultures, cells appeared to spend approximately the same amount
of time in each stage of mitosis. In EB1-depleted cells, however, there is an
accumulation at metaphase (~40% compared with 22% in controls) and in
telophase (~43% compared with 38% in controls). These data suggest that
inhibition of EB1 activates the spindle checkpoint. Further work will be required
to understand potential checkpoint activation in response to loss of EB1 function,
perhaps by live cell imaging (Rogers, 2002).
During normal mitosis, the mitotic spindle positions itself at the geometric center
of the cell. In S2 cells lacking EB1, however, the spindle is frequently

Using time-lapse spinning disk confocal microscopy.35 µm ± 0.and GFP RNAi-treated cells. During interphase of cycle 12. documented progression.42 µm ± 0. the spindle center was determined by measuring the distance between the two poles in cells that had their mitotic spindle aligned parallel to the coverslip. because thousands of synchronous spindles that divide within a two-dimensional plane can be readily observed by confocal microscopy. it is concluded that loss of EB1 function causes mispositioning of the mitotic spindle (Rogers. In contrast. respectively. the average offset distances between the centroid and the spindle center were 0. because preblastoderm embryos are not governed by the same mitotic checkpoint mechanisms as differentiated cells. extended live cell imaging of the spindle proved technically difficult. From these data. the Drosophila synctitial blastoderm was used as a model. duplicated . the average offset distance in EB1-deficient cells was significantly greater. In control embryos. In untreated and GFP dsRNA-treated cells. Furthermore. 2002). the effects were observed of EB1 inhibition on mitotic spindle formation. To quantitate this effect.15. the later stages of mitosis can be observed after treatments that might otherwise induce mitotic arrest by activation of the spindle checkpoint. the centroid of the cell ws calculated and the offset distance between the cell centroid and the spindle center was determined for untreated cells and for EB1.93 µm ± 0. dynamics of the mitotic spindles followed a wellcharacterized. Then. 2002). and chromosomal segregation (Rogers.mispositioned.57. spindle elongation. Although the Schneider cell system provided a convenient method to generate loss-of-function phenotypes for EB1. anti-EB1 antibodies were microinjected into transgenic preblastoderm embryos expressing GFP-tubulin.17 and 0. 1. To study the role of EB1 in spindle dynamics in vivo. To study the role of EB1 in spindle dynamics.

03 µm/s until reaching a separation of ~12 µm. spindles further elongate at a rate ~0. with the most severe defects centered around the injection site. The exact . EB1 antibodies were microinjected into Drosophila embryos expressing GFP-tubulin. As the cells progress to metaphase. Given the general similarity in phenotypes observed with antibody microinjection and RNAi. spindles elongate at a rate of ~0. 2002). A few minutes after chromosomes congress to the metaphase plate. the possibility cannot be excluded of effects manifest through other polypeptides (e. This phenotype is similar to that produced by RNAi of EB1 in S2 cells. the weakly reactive 75-kD polypeptide observed after long exposure of immunoblots). After nuclear envelope breakdown of cycle 12. it is believed that the antibodies are manifesting their effects through EB1. Upon anaphase onset. it becames readily apparent that the spindles closest to the injection site have fewer microtubules and have a shorter pole-to-pole distance than controls. the nuclear envelope break down and the nuclear space is invaded by microtubules emanating from opposite poles.5 µm.07 µm/s until reaching a maximal length of ~16. These microtubules form attachments either with chromosomes to form kinetochore fibers or intercalate with microtubules of opposite polarity to form interpolar bundles.centrosomes move to opposite sides of the nucleus to positions separated by ~120°. The pole-to-pole distances are highly reproducible in spindles throughout cycle 12.g. Upon entry into prometaphase. However. The injected antibodies produced a readily apparent gradient of effects on spindle structure and behavior. the length of the spindle is ~8 µm. As these embryos approach cycle 12 mitosis. To investigate whether EB1 plays a role in mitosis in this system. These measurements are in close agreement with a previous description of Drosophila embryo spindle dynamics (Rogers.. the spindles transits to anaphase and sister chromosomes segregate to opposite poles to complete mitosis.

EB1 antibodies and rhodamine-labeled histones were coinjected into embryos expressing GFP-tubulin to simultaneously observe the behaviors of chromosomes and microtubules. In addition to reduced rates of elongation. Spindles further from the injection site exhibited a pole-to-pole distance that more closely resembled controls.5 µm) at metaphase. In control embryos. Regions of these embryos distal to the injection site supported formation of morphologically normal spindles that progress through mitosis similar to controls (Rogers. Observation of these defects over time revealed that spindle structure was dynamic and these frayed and monopolar spindles could sometimes correct themselves and complete mitosis. During the prophase-to-metaphase transition.015 µm/s) and achieve a shorter length (8. spindles elongate threefold slower (~0. fluorescent . 2002). 2002). but also frequently displayed structural defects such as frayed and monopolar half spindles that had both centrosomes present at a single pole. At anaphase. it was speculated that proper chromosome segregation could be affected as well.2 ± 0. spindles elongate twofold slower (~0.mechanism of the antibody-induced defect is not known. although the similarity to the RNAi phenotype makes gives rise to the suspicition that the antibody injection leads to a loss of EB1 function (Rogers. 2002). If interference with EB1 activity disrupts normal spindle elongation at anaphase. The effects of EB1 inhibition on spindle elongation were quantitated by measuring the pole-to-pole distances of spindles proximal to the injection site over time.01 µm/s) and elongate to a maximal length 40% less than controls (9.1 ± 0. To test this hypothesis.6 µm). spindles proximal to the injection site exhibit a striking overall reduction in associated microtubules and fail to form normal interpolar microtubule bundles or a midbody at the end of anaphase (Rogers.

and CG2955.6%) or stretched (31%) chromosomal masses that fail to segregate and decondense midway between the poles at the end of mitosis. chromosome congression to the metaphase plate. The most extreme defect observed is complete inhibition of chromosomal segregation. Taken together. CG18190. More deleterious effects are produced when the chromosomes begin to segregate but fail during anaphase. Injection of EB1 antibodies. termed here Drosophila EB1) exhibits a higher degree of sequence identity throughout its length to both human EB1 (52%) and Saccharomyces cerevisiae Bim1p (33%). producing bilobed (8.histones incorporated into chromatin and allowed observation of chromosome condensation at prometaphase. leading to the formation of a tetraploid nucleus in between two spindle poles. EB1 promotes microtubule dynamics in Drosophila cells To begin analysis of EB1 function in Drosophila. 2002). Residues 1-134. 2001). The Drosophila genome contains four predicted gene products that encode proteins with a high degree of sequence similarity (>40%) to human EB1: genes CG3265.5 kD) with a similar domain organization to human EB1 and Bim1p. making it the most likely orthologue. the fly genome was examined for genes that exhibited homology to human EB1 (MAPRE1) (Su. The mildest defect caused by EB1 antibody injection was the generation of lagging chromosomes during anaphase (30%). CG15306. which have the highest degree of sequence conservation among EB1 . and sister chromatid separation and segregation to each pole during anaphase. these results from microinjecting anti-EB1 antibodies into preblastoderm embryos indicate that EB1 plays a crucial role in mitotic spindle formation and elongation and is needed for the proper segregation of mitotic chromosomes during anaphase (Rogers. One gene (CG3265. disrupts chromosome segregation and produces a range of phenotypes. This gene encodes a predicted protein of 294 residues (32. however.

Sentin is a novel EB1 cargo protein. the orthologue of XMAP215 microtubule polymerase. resulted in an increase in microtubule pausing and led to the formation of shorter spindles. recruit multiple cargo proteins. In Bim1p. constitute the domain of the protein implicated in microtubule binding (Juwana. several genes were identified whose knockdown leads to the shortening of the metaphase spindle. was named 'Sentin' from the Japanese word Sentan (tip).family members. 2002). and the metaphase spindle length was 72% of the control. Among them. Residues 129-212 are enriched in serines and prolines and hence may be unstructured. Sentin's association with EB1 is critical for its plus end localization and function. and are critical for making dynamic microtubules in vivo. These results indicate that EB1 promotes dynamic microtubule behavior by recruiting the cargo protein Sentin and possibly also a microtubule polymerase to the microtubule tip (Li. Furthermore. without displacing EB1 from growing microtubules. 2000). one uncharacterized gene. After RNAi treatment. Sentin levels were reduced by 90%. similar to EB1 depletion. In a genome-wide RNAi screen using S2 cells. the EB1 phenotype rescued by expressing an EBN-Sentin fusion protein in which the C-terminal cargo-binding region of EB1 is replaced with Sentin. Centrosome/centriole-free spindles . Knockdown of Sentin attenuates plus end accumulation of Msps (Mini spindles). CG9028/ssp2. the COOH-terminal coiled-coil domain binds to Kar9 (Miller. and residues 213-273 are predicted to form a coiled coil. Sentin depletion in Drosophila S2 cells. Highly conserved EB1 family proteins bind to the growing ends of microtubules. it is unclear how these master regulators of microtubule plus ends promote microtubule dynamics. and it may mediate protein-protein interactions in other species as well (Rogers. 1999). 2011). which was similar to EB1 or XMAP215Msps RNAi. However.

2009). the known structural changes of the plus end can be made by the EB1 protein alone. so that tubulin dimers are favorably added to or removed from the end. Furthermore. Thus. EB1-Sentin makes microtubules dynamic by further recruiting XMAP215Msps polymerase to the tip. This model is consistent with the phenotypic similarity among EB1. Sentin catalyses the supply and removal of tubulin dimers at the microtubule plus ends. However. In the third model.constructed using Sak (Plk4) RNAi were also shorter in the absence of Sentin than in the control cells. closing the sheet or promoting a specific lattice configuration. and XMAP215Msps and also the observation in Xenopus laevis egg extracts in which the EB1 depletion phenotype is rescued by overexpression of XMAP215 (Kronja. . which is similar to the case of XMAP215 polymerase. 2011). a series of truncation and gene fusion experiments raised an intriguing possibility that Sentin is the dominant cargo of EB1 for microtubule dynamics promotion in S2 cells. e. 2011). Sentin. the formation of shorter spindles is likely to be caused by the presence of shorter microtubules in the Sentin-depleted spindles (Li.g. However. In the second model.. The first possible model that how might Sentin promote plus end dynamics is that the EB1-Sentin complex alters the structure of the plus end of microtubules. Studies have identified Sentin as an EB1 cargo protein. it is not ruled out that the cytoplasmic pool of other EB1 cargo proteins is also required to suppress microtubule pausing (Li. These two activities may be responsible for the antipause activity of the EB1Sentin complex observed in vivo. This idea is consistent with the fact that no other known cargo proteins phenocopy EB1 as closely as Sentin in S2 cells and that EB1 protein alone does not show growth-promoting activity in vitro in several studies.

The similarity of three of them to EB1 also is extended to the Cterminal region. including flightlessness and uncoordinated movement. Among them. although their expression is rather limited. electrophysiological measurements indicated that the auditory chordotonal organs show a reduced response to sound stimuli. DmEB1 plays a crucial role in the functional and structural integrity of the chordotonal mechanosensory organs in Drosophila (Elliott. Three mutant alleles of . is ubiquitously expressed and has essential functions during development. In Drosophila. Hypomorphic DmEB1 mutants show neuromuscular defects. 2005) Drosophila is used as a model system to examine in vivo roles of one of the EB1 homologues in a developmental context. These defects can be partly explained by the malfunction of the chordotonal mechanosensory organs. Consistently. proteins encoded by up to six genes share some homology to EB1 in the N-terminal region (microtubule binding domain. One of the multiple Drosophila EB1 homologues. In fact.Crucial role of EB1 in the integrity of chordotonal mechano sensory organs of Drosophila EB1 is a conserved microtubule plus end tracking protein considered to play crucial roles in microtubule organization and the interaction of microtubules with the cell cortex. Other EB1 homologues also are expressed during development. Drosophila EB1 protein is ubiquitously expressed during Drosophila development. without any general cell division defects. The internal organization of the chordotonal organs also is affected in the mutant. Drosophila EB1 is the most similar to human EB1. Despite intense studies carried out in yeast and cultured cells. the role of EB1 in multicellular systems remains to be elucidated. DmEB1 is enriched in those regions important for the structure and function of the organs. DmEB1. Therefore.

it is likely that the phenotype observed in EB1 mutants represents a subset of EB1 functions in vivo. Therefore. Consistently. they show uncoordinated body movements when correcting the body position after being overturned. It is also possible that other EB1 homologues may have redundant functions that compensate for loss of EB1 in Drosophila. studies on semilethal alleles of EB1 revealed defective neuromuscular functions. it is rather puzzling at first sight that the EB1 mutants showed limited defects without significant abnormalities in universal cell functions. Moreover. rare . Therefore. However. Nevertheless. the DmEB1 gene has a unique and essential function that is not complemented by the other homologues during Drosophila development. Transgenic wild-type EB1 genes expressed from the ubiquitin promotor fully rescues these DmEB1 mutants. These adult flies are flightless and hold their wings in the wrong position when resting. Moreover.Drosophila EB1 were identified. one of which is lethal and the other two are semilethal. Previous studies using RNA interference (RNAi) or antibody injection indicated that DmEB1 is required for the regulation of microtubule dynamics and spindle organization/positioning. Inability to complement the DmEB1 defects could be due to differences in expression or protein activity of the other EB1 homologues (Elliott. the hypomorphic mutations uncovered the neuromuscular involvement of EB1 without interference by more universal cellular roles (Elliott. 2005). Although the EB1 alleles do not show significant cell division or morphological defects. EB1 protein has a large maternal contribution that may mask the requirement in early development. Therefore. 2005). it should be emphasized that the EB1 mutations examined in this study are hypomorphic and a low level of active protein produced in the mutants may be sufficient to support spindle formation.

2005). 2005). and via transcellular attachments to the actin-based rods of the scolopale. unidentified defects (Elliott. at their base to a ciliary rootlet. a spindle-shaped structure encasing a sensory cilium. Disruption of any of these linkages could result in a reduced response (Elliott.escapers of a stronger allele show much more severe body coordination defects (Elliott. Therefore. including cap cells. These coincide with structurally important regions for chordotonal functions. They include (1) anterior . mutants completely lacking chordotonal organs show better viability than the DmEB1P homozygotes. ligament cells. Mechanotransduction typically requires intact mechanical linkages between stimulus-delivering structures and neuronal transducing elements. and scolopale cells. These behavioral phenotypes of DmEB1 mutants share some characteristics with the phenotypes of mutants defective in chordotonal sensory organ function. Each sensory unit of a chordotonal organ is comprised of one or a few ciliated neurons and several supporting cells. indicating that the EB1 mutants have additional. in part. explain the EB1 mutant defects. 2005). it is concentrated at high levels in subregions of supporting cells in the chordotonal organs. rather than EB1 distributing uniformly to all cells in the chordotonal organs. DmEB1P mutant is indeed defective in the function of auditory chordotonal organs. Sensory cilia are connected at their tips to the extracellular dendritic cap. The immunolocalization study of EB1 indicated that. Chordotonal organs are mechanosensory organs that act as stretch receptors and also form the fly's auditory systems. However. The central structure is the scolopale. malfunction of chordotonal organs can. These structures are connected to other body structures through cap cells and neurons/ligament cells.

one possibility is that this misalignment may represent an underlying weakness of cell structures such as the area supporting inner segment of neuron dendrites. scolopale structures. although all cells are likely to express and require it at some level. and ciliary rootlets. The loss of EB1cause the defects in function and organization of chordotonal organs at the molecular level. 2005). These cell structures probably require a high level of EB1 and are sensitive to a reduction of the protein level in the mutant. the defects of other EB1-rich regions may well contribute functional and structural defects of chordotonal organs in the mutant (Elliott. This misalignment is often associated with unusually stretched dendrite morphology. basal bodies. 2003) the EB1 homologue is localized to the flagellar tip and implicated in intraflagellar transport in (Elliott. Any defects in spindle positioning or orientation disrupt asymmetric divisions and result in a . It also should be noted that EB1 does not have significant concentration at ciliary endings. Immunostaining of mutant chordotonal organs in the abdomen revealed that. (3) regions of cells surrounding the inner segment of neuron dendrites. (2) scolopale region. such as cilia. Electron microscopy of mutant chordotonal organs revealed ultrastructures with normal morphology. In addition. RNAi in Drosophila revealed that EB1 plays roles in regulating the mitotic spindle organization and spindle positioning/orientation. Comparing the mutant phenotype with the immunolocalization results. 2005). although the overall arrangement of the chordotonal organs and the number of cells are not altered. the alignment of the dendrites is disrupted. dendritic cap. although inChlamydomonas (Pedersen. Any defects in chromosome segregation may lead to a failure to produce the sufficient number of cells comprising the chordotonal organs.ends of cap cells that are in contact with the body walls. and (4) part of the ligament cells.

However. which localizes to the cell cortex. chordotonal organs require robust mechanical linkages between stimulus-delivering structures and neuronal transducing elements to sense tensions effectively (Elliott. interacts with EB1 and is shown to be required for chordotonal organ integrity. Microtubule plus end tracking proteins are considered to play . it is possible that DmEB1 may link microtubules to the cell cortex via interactions with Short stop. Cells comprising chordotonal organs are highly polarized and have specialized cytoskeletons both molecularly and morphologically.failure of correct fate determination among cells comprising chordotonal organs. Furthermore. 2005). The chordotonal phenotype of the short stopmutant is similar to defects seen in the DmEB1 mutant. 2005). 2005). Therefore. Immunofluorescence microscopy using antibodies against α-tubulin and Futsch (the MAP1 homologue) or electron microscopy could not resolve the precise defect of the cytoskeleton (Elliott. Instead. no general cell division defects or significant alteration of the cell numbers were observed in chordotonal organs of the mutant (Elliott. observations on mutant chordotonal organs are consistent with roles of EB1 on microtubule cytoskeleton. DmEB1 and homologues in other organisms are shown to regulate interphase microtubule dynamics and thought to be involved in interactions between microtubule plus ends and the cell cortex. they may be particularly sensitive to the reduction in EB1 activity. The actin-microtubule linker Short stop. therefore. chordotonal organs are highly polarized and are required to withstand strong mechanical forces. Kinetochore and EB1 associated basic protein (Kebab) Microtubule plus ends are dynamic ends that interact with other cellular structures. Although the linkage between microtubules and the cell cortex is important for most cell types.

Generation of mutants lacking the kebab gene indicated that Kebab is dispensable for viability and fertility (Meireles. Several studies have successfully identified EB1-interacting proteins by mass-spectrometry after pull down using bacterially produced EB1 protein. and a coiled-coil domain. CG31672). The protein has a domain most similar to the atypical CH domain of Ndc80. it associates with microtubules in central-spindle and centrosomal regions. In vitro expression cloning was originally developed.important roles in the regulation of microtubule plus ends. through in vitro expression screening for EB1-interacting proteins. Studies report the identification of a novel Drosophila protein. One drawback of this approach is that the chance of a protein being identified depends on its level in a particular tissue or cell line. The localisation to kinetochores depends on microtubules. 2011). This study identified a novel EB1-interating protein. which was called Kebab (kinetochore and EB1 associated basic protein. EB1 regulates microtubule plus ends through interaction with multiple proteins. using random cDNAs from Xenopus eggs. Kebab was identified by in vitro expression cloning. Kebab fused to GFP shows a novel pattern of dynamic localisation in mitosis. Recent studies revealed that EB1 is the central regulator for microtubule plus end tracking proteins by recruiting them to microtubule plus ends through direct interaction. Depletion of Kebab in cultured cells by RNA interference did not show obvious defects in mitotic progression or microtubule organisation. The interaction with EB1 is mediated by two SxIP motifs but is not required for the localization. Kebab. In telophase. This study looked for substrates of a kinase by examining the shift in gel . It was later adapted for use with a collection of annotated unique Drosophila cDNAs. It localises to kinetochores weakly in metaphase and accumulates progressively during anaphase. which shows a dynamic localisation to kinetochores and microtubules in mitosis.

Kebab localises to kinetochores during mitosis. Kebab is not colocalised with CenpA. have been reported to progressively accumulate on centromeres during anaphase. The second interesting property of Kebab is that microtubules are required for its kinetochore localisation.mobility after kinase reaction. It is possible that Kebab recruitment to kinetochores may be somehow linked to CenpA loading. This property is unusual but not unique among kinetochore proteins. Interestingly. CenpC and Mis18. generally they do not increase in intensity during anaphase. rather than other kinetochore proteins. suggests it localises to outer kinetochores. which is coupled to microtubule polymerisation. as long as cDNA has been isolated from some tissues (Meireles. The relative position of Kebab to the Drosophila CenpA orthologue Cid. It is still not understood why this occurs during anaphase. 2011). The advantage of this approach over a massspectrometry based one would be that low abundance proteins or those expressed in specific cell types have equal chance of being identified. rather is localised outside of CenpA. including CenpA. 2011). It was found to localise to only one of the sister kinetochores moving away from poles. a Histone 3 variant. These proteins interact with each other to define the centromere identity through loading of CenpA. Alternatively. In contrast. The most notable feature of Kebab is that it progressively accumulates on kinetochores during anaphase. or the dose of CenpA on centromeres. Although this behaviour is unusual among kinetochore proteins. Although all kinetochore proteins require CenpA for their localisation. EB1 localisation to kinetochores in PtK1 cells was previously reported to depend on microtubules. This method was further adapted for a pull down assay to identify interacting proteins. on DNA. Kebab may be regulated by a previously unknown mechanism (Meireles. a group of centromere proteins. Kebab localisation appears to be .

other types of proteins are known to change localisation during mitotic progression.symmetrical in metaphase and become more prominent when microtubules are depolymerising in anaphase. The association with microtubules becomes more prominent in telophase. suggesting interaction with EB1 is not essential for localisation. The localisation of Kebab dynamically changes during mitotic progression. The SxIP motif is a linear motif found in many EB1 binding proteins. Kebab can directly bind to EB1 in vitro. In either case. and stabilisation of the spindle midzone in telophase. Nevertheless. 2011). Dynamic localisation of Kebab may subtly influence a change in behaviour of kinetochore or spindle microtubules (Meireles. Kebab may recognise kinetochore-associated microtubule plus ends regardless of the polymerisation state. Consistently it was found that EB1 depletion did not disrupt the localisation of Kebab. For example. Other kinetochore proteins such as Ska1 and Ajuba are also reported to depend on microtubules for their localisation. Kebab starts localising to spindle microtubules or centrosomal regions. Alternatively. The mechanism and significance of this microtubule dependency are still under speculation. 2011). It is possible that EB1-independent localisation masks the . This change in localisation is considered to be crucial for the change in kinetochore behaviour at the onset of anaphase. the chromosomal passenger complex localises to centromeres/kinetochores until metaphase and relocates to microtubules in the spindle midzone at the onset of anaphase. Mutations in both SxIP did not abolish the localisation of Kebab. This changing pattern of localisation is unique among previously reported kinetochore proteins or microtubule-associated proteins. In late anaphase. Interaction with EB1 is mediated by two SxIP motifs located near the CH domain. it is a very interesting and unusual property of a kinetochore protein (Meireles. Kebab is transported to kinetochores along microtubules.

2011). there may be other proteins that function redundantly with Kebab. Ndc80. These consist of a diverse array of proteins. For example. microtubule bundling can be achieved by many proteins or protein complexes which contain multiple microtubule binding sites. 2011). It is likely that many structurally distinct proteins can function redundantly to regulate microtubules. and was only recognised after the crystal structure was determined. The CH domain of Ndc80 is quite distinct from typical CH domains found in other proteins. This domain of Ndc80 is considered to be the microtubule binding domain. Alternatively EB1 interaction may be important for Kebab function rather than the localisation.EB1 dependent localisation in a specific location. 2011). Further studies are needed to clarify the significance of EB1 interaction (Meireles. with only a small minority containing known microtubule binding motifs. Ndc80 is one of the critical proteins which connect kinetochores to microtubules. Kebab contains a domain which is most similar to the atypical CH domain of another kinetochore protein. No obvious functions have been revealed by RNAi or generation of null mutants. such as microtubule regulation. Hundreds of proteins in each cell type can bind to microtubules. It is a challenge in biology to understand a system that involves many redundancies. . Future identification of proteins that have overlapping function with Kebab will shed light on the function and regulation of Kebab protein in mitosis (Meireles. Although there are no obvious paralogues in the Drosophila genome. and collectively determine their behaviour. The discovery of the second member of this atypical CH domain group may shed light on how the essential kinetochore protein Ndc80 interacts with microtubules (Meireles.

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