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The Cytoskeleton and Cellular Movement: Microfilaments and intermediate

filaments
Chao Chung-Faye ( 趙壯飛 )
DDS, MS and PhD
Professor, Executive Director, Institute of Life Sciences
National Defense Medical Center

I. Cell Shape: Isolated single cells in suspension condition were


mostly present round in shape. Once the cells attached to matrix or
contact to gather, they were formed a different shape depend on their
specific phenotype,. such as neuron cell, fibroblast, different
epithelial cells. All the cells shape were maintained by their specific
arrangement of their cytoskeleton.

• Cell movement: It include beating of the cilia and flagella, contraction of


muscle cells, the movement of chromosomes, and the migration of the cells
along a substratum.

• Intracellular trafficking: Macromolecules and organells movement.


Cell Motility and Shape I:
Actin-Myosin and Intermediate Filaments
Actin Structure

1. A major component of cytoskeleton, the actin is the most abundant intracellular protein in eukaryotic
cells and is highly conserved: A globular protein with 42kd in Molecular weight. A monomeric subunit
as G-protein. The sequence of actins from amebas and from animals are identical at 80% percent of the
positions.
2. The various actin isoforms exhibit minor sequence differences but generally perform different functions:
4-α. isomer- associate with contractile structure, β actin-leding edge where actin fliaments
polymerization and γ actin – for the filaments for stress fiber

4-α. Isomer:
Smooth muscle cell
Skeletal muscle cell
Non-smooth muscle cell
Amino acid sequence homology among SM22 proteins from different species
Actins R62D, G13R, VP.C, and G150P do not colocalize with
cellular F-actin structures. NIH3T3 cells were transiently
transfected with expression plasmids encoding FLAG-actin or
mutant derivatives together with the nuclear-localized LexA
derivative NLexA as transfection marker. Transfected cells
were formaldehyde fixed and stained as described in
MATERIALS AND METHODS. Left, transfected actins
(anti-FLAG; green). Right, merge of transfected actins (anti-
FLAG; green), total F-actin (rhodamine-phalloidin; red) and
transfection marker (anti-LexA; blue). Bar, 50 µm. 1

R:Arginin D:Asppartic acid


G:Glycin
P: Prolin
Actins R62D, G13R, VP.C, and G150P can
be quantitatively removed from cells by
detergent extraction. NIH3T3 cells were
transiently transfected with expression
plasmids encoding FLAG-actin or mutant
derivatives as in Figure 1, but the cells were
subjected to brief extraction with 0.5% Triton
X-100 before fixation and staining. Left,
transfected actins (anti-FLAG; green). Right,
merge of transfected actins (anti-FLAG;
green), total F-actin (rhodamine-phalloidin;
red) and transfection marker (anti-LexA;
blue). Bar, 50 μm.
Actins V159N and S14C stabilize F-actin and
activate SRF. (A) Actins V159N and S14C
colocalize with endogenous F-actin structures.
NIH3T3 cells were transiently transfected with
expression plasmids encoding FLAG-actin or
mutant derivatives and subjected to brief extraction
with 0.5% Triton X-100 before fixing and staining
as in Figure 2. The Figure shows a merge of
transfected actins (anti-FLAG; green), total F-actin
(rhodamine-phalloidin; red), and transfection
marker (anti-LexA; blue). Bar, 50 µm. (B) Actins
V159N and S14C accumulate preferentially in the
Triton X-100-insoluble fraction. Extracts of cells
expressing the indicated FLAG-tagged actin
mutants were prepared and separated into 100,000-
g supernatant (S) and pellet (P) fractions as
described in MATERIALS AND METHODS;
equal amounts were separated by SDS-PAGE and
FLAG-actin in each fraction was detected by
immunoblotting with anti-FLAG antibodies. (C)
Actins V159N and S14C copolymerize with wild-
type actin and decrease its detergent solubility.
Extracts were prepared from cells expressing the
V:Tyrsine indicated actins, or the catalytic domain of LIMK1,
N: Asparagin together with HA-tagged wild-type actin and
S:Serin fractionated as in B before detection of HA-actin in
C:Cystein each fraction by immunoblotting with anti-HA
SRF: serum response factor antibodies

. (D) Expression of actins V159N and S14C increases F:G-actin ratio. Cells were transfected with expression plasmids encoding the
indicated actins and fixed and stained for the FLAG epitope and either F-actin, with phalloidin, or G-actin, with DNase I. The levels of F-
actin or G-actin in the transfected population were then quantitated relative to the untransfected population by using FACS, as described
in MATERIALS AND METHODS. Bars represent the increase in mean relative F-actin (±SEM, n = 4) or G-actin (± half range, n = 2)
contents. (E) Expression of actins V159N or S14C activates SRF in the absence of extracellular signals. NIH3T3 cells were transfected
with expression plasmids encoding the indicated FLAG-tagged actins together with the SRF reporter gene 3D.ALuc as described in
MATERIALS AND METHODS. After serum stimulation, luciferase activities were measured and expressed relative to activation by
SRF-VP16, taken as 100. Data show mean of at least four independent experiments with SEM indicated by the bars.
3. F-actin is a helical filamentous polymer of G- actin subunits all oriented in the same direction

Each actin molecule contains a Mg2+ ion complexed


With either ATP or ADP. Thus there are four states
of actin: ATP-G-actin, ADP-G-actin, ATP-F-actin and
ADP-F-actin.
4. Actin filaments are organized into bundles and network by variety of bivalent cross-link proteins:
Many actin cross-link proteins belong to the calponin homology-domain superfamily (which has a pair
of actin-binding domains whose sequence is homologous to that of calponin.
The Dynamics of Actin Assembly

1. Within cells, the actin cytoskeletone is dynamic, with filaments able to grow and shrink
rapidly. Polymerization of G-actin in vitro is marked by a lag period during which
nucleation occurs. Eventually, a polymerization reaction reach a steady state in which
the rates of addition and loss of subunits are equal.
2. The concentration of actin monomers in equilibrium with the actin filaments is the
critical concentration . At a G-actin concentration above Cc, there is net growth of
filaments; at concentrations below Cc, there is net depolymeriztion of filaments.
3. Actin filaments grow considerably faster at their (+) end than at their(-) end, and the Cc
for monomer addition to the (+) end is lower than that for addition at the (-) end.
actin cytoskeleton and its capacity to assemble and disassemble rapidly
and at the appropriate location within the cell, the so-called actin
treadmill
4. The assembly, length, and stability
of actin filaments are sever filaments or
cap the ends or both. These proteins are
in turn regulated by various
mechanism.

A. Thymosin β4: It considered to be


the main actin-sequestering protein in
cells. It binds ATP-G-actin in a 1:1
complex. The binding of thymosin β4
blocks the ATP-binding site in G-
actin, thereby preventing its
polymerization.

B. Profilin promote actin assembly:


1. Acting as a nucleotide-
exchange factor
2. To assist in the addition of
monomers to the (+) end of an actin
filament.
3. It can interact with
membrane components taking part in
cell-cell signaling.
Cofilin in lamellipodia
Actin filaments nucleation and web
formation by the ARP complex in
lamellipodia

ARP complex: green


Actin: red with phallodin
Myosin-Powered Cell Movement:

1. All myosin isoforms can interact with actin filaments through their head domains, but
their cellular roles differ, depending on their tail domain.
2. Movement of actin filaments by myosin can be directly monitored in the sliding-filament
assay
3. Myosin-bound vesicles are carried along actin filaments:
A. vesicle trafficking ( Myosin I, V and VI):
B. Cytoplasmic streaming (Myosin XI):
II. Smooth muscle cells contraction is calcium-dependent activation of myosin II
III. SMC and non-SMC cells contraction is signal induced activation of myosin II by Rho
kinas
Actin and Myosin in non-muscle cells

I. Organized bundles of actin filaments maintain microvilli structure:


II. Stress fibers permit cells to attach to the surface:
III. Actin microfilaments are found in the cortex of many nonmuscle cells:
Cell Locomotion

I. Cell movement coordinates force generation with cell adhesion:


membrane extension Cell substrate adhesion
Cell body translocation Breaking cell attachment
II. Ameboid movement entails reversible Gel-Sol transitions of actin networks
III. External signals and various signaling pathways coordinate events that lead to cell
migration:

A. Activation of filopodia, membrane ruffles, and stress fibers by growth factors


Focal contacts and stress fibers in a cultured fibroblast.
The dramatic effects of Rac, Rho, and CDc42 on actin organization in fibroblast
B. Steering of migration cells by chemotactic molecules:
C. Coincident gradients of chemoattractants, activated G proteins and Ca++
Neurophil polarization and chemotaxia:

The pipette tip at the right is leaking a small


amount of the peptide formyl-Met-Leu-Phe
Intermediate filaments: It found in nearly all animal cells but not in the plants and fungi.
The association of intermediate filaments with nuclear and plasma membrane suggests that
their principle function is structural. Intermediate filaments are strong and ropelike
structure. It can strength the cell against mechanical stress.
Structure of Intermediate filaments:
Nucelar lamin: There are three isoforms within the nuclear and the nuclear
envelope is supported by the lamin filament
Keratin:
Keratin
1. Central rod domain

K14 50kDa
K5 58kDa

2. Neutral-basic keratins : K1~K8 PI>6 53~67 kDa


Acidic keratins : K9~K20 PI<5.5 40~63 kDa
3. Pair

4. Embryo development K14


Epithelium differentiation K14
Cornified layer

Granular layer

Upper spinous layers

Lower spinous layers

Basal layer

Lamina lucida
Lamina densa
Anchoring fibrils
Markers of Terminal Differentiation

Stratified layer Filaggrin


Granular layer Profilaggrin and loricrin
Upper spinous layer K1, K10, and involucrin
Lower spinous layer K1, K10, and involucrin
Basal layer K5 and K14
Down regulation of Keratin 14 during terminal differentiation

Stratum corneum
Keratin 1/10
Stratum granulosum

Stratum spinosum
Stratum basale Keratin 5/14
(type I acidic keratin
Dermis polypeptide of 50 kDa)
NF-kappa B in epidermis(1)

nuclear
cytosol

inhibit expression normal over expression

Seitz et al. 1998.


The relationship of NF-κB with K14 promoter(1)

Ma et al. 1997.
K14 全長
VECTOR VECTOR

NE Negative element

NE1 NE2
EΠ ElementΠ

EΠ1 EΠ2

SmaΙ SmaΙ

Enhancer
vector EΠ1 enhancer EΠ enhancer
vector vector
K14P-2000
-2000
Luc.

K14PΔ-391~-321
-2000 -391 -321
Luc.

K14PΔ-367~-343
-2000
-367 -343 Luc.

K14P-280
-280
Luc.
0
5
10
15
20
25
30
pG
L3
K1
4P
K1 -2
4P 00
-2 0
K1 00
0+
4P p6
K1 Δ 5
4P -36
Δ 7~
-3 -3
67 43
~-
K1 34
3+
Cotransfection assay results

4P p6
K1 Δ 5
4P -3
Δ 91
~-
-3 32
91 1
~-
32
1+
p6
5
K1
4P
K1 -2
4P 80
-2
80
+p
65
TPA

p65
- +

K14 promoter
-405 -380 -344 -280
Vimentin: Many cells of mesenchymal origin like fibroblas

Figure 5.
Nestin expression and the assembly state of
vimentin in MDBK cells. (A and B) All cells
display a filamentous vimentin pattern
(VIM, red), but fewer are positive for nestin
(NES, green), as determined by double
indirect immunofluorescence. During
mitosis, cells exhibiting brighter nestin
fluorescence contain a rather punctate
vimentin and nestin pattern (C and D),
whereas cells expressing no or low levels of
nestin have a filamentous vimentin network
(E and F). Mitotic chromosomes are stained
with toto-3 (blue). Bar, 10 μm.
FTCD: forminaotransferase and cyclodeaminase: Golgi protein
Figure 3.
Phosphorylation of vimentin at ser-55 is
required for the nestin-mediated
disassembly of IFs during mitosis. Wild-type
vimentin (WT) or either the ser-55:ala (S55A) or thr-
457:ala/ser-458:ala (S458A) mutant vimentins were
transfected individually or together with nestin in
MCF-7 cells. Subsequently, the assembly state of
vimentin was examined by indirect
immunofluorescence with a rabbit antibody directed
against vimentin (red) and a monoclonal antibody
directed against rat nestin (NES, green). In interphase
cells, typical vimentin IF networks assembled from WT
vimentin alone (A) or from WT vimentin and nestin as
demonstrated by double-label immunofluorescence (B
and C). During mitosis, all three forms of vimentin,
when expressed individually, retained their
filamentous networks (D–F). However, when vimentin
was coexpressed with nestin, IFs formed with WT
vimentin/nestin (G and J) or with thr-457:ala/ser-
458:ala-vimentin/nestin (I and L) were disassembled
into punctate and diffuse structures, whereas IFs
assembled with ser-55:ala-vimentin/nestin remained
intact (H and K). Images G and J, H and K, and I and
L are pairs of the same cells using double
immunofluorescence. Mitotic cells were identified by
the presence of condensed chromosomes (blue). (M)
Summary table of the quantitative results of singly and
doubly transfected mitotic MCF-7 cells. Bar in
interphase (A–C) and mitotic (D–L) cells, 10 μm.
Figure 6.
Nestin-specific siRNA prevents the disassembly
of vimentin IFs in mitotic BHK-21 cells.
Interphase cells display filamentous vimentin
(VIM, red) and nestin (NES, green) networks
that are very similar to each other (A–C,
double-label and merged images). After cells
were treated with nestin siRNA, nestin
fluorescence was greatly reduced or
nondetectable in many cells. Reduction of nestin
expression had no detectable effect on the
expression or the organization of endogenous
vimentin (D–F, double-label and merged
images). Untreated mitotic cells had both
vimentin and nestin in a punctate pattern (G–I).
In mitotic cells, in which the nestin signal was
greatly reduced by nestin siRNA, the vimentin
remained in a filamentous pattern (J and K).
The mitotic state of the cells was identified by
condensed chromosomes in blue. (L)
Immunoblotting analysis of vimentin and nestin
in cell lysates derived from cells untreated (−) or
treated (+) with luciferase GL2 siRNA (GL2) or
nestin siRNA (NES). Bar, 10 μm.
Desmin: muscle
4 Immunofluorescence analysis of desmin expression in the SW13 (vim-) cells transfected with A, pDesC
containing normal desmin cDNA sequence, and B, pDesV containing the patient's mutant cDNA. Bars, 20 m.
Normal desmin formed an abundant and well-structured network, while desmin carrying the R406W mutation
was not capable of forming a filamentous network and instead aggregated into desmin-positive granules.
GFAP: glia
fibrose acidic
protein

Newly generated cells with neuronal characteristics in the adult macaque monkey brain. (A and B) Immature neurons expressing TuJ1
(red) are distributed in the SVZ along the lateral and ventrobasal walls of the lateral cerebral ventricle. BrdU-labeled nuclei (greenish-
yellow; arrows) indicate that these are newly generated cells. (A) Immature morphology and the colocalization of BrdU within the nuclei
of TuJ1-labeled neuroblasts. (B) Neuroblasts in the SVZ coalesce into migratory chains that extend via the rostral migratory stream to the
olfactory bulb. Migrating neuroblasts were not observed in the subcortical white matter. Ependymal and astrocytic cells are indicated by
GFAP (blue). LV, lateral ventricle. (C and D) An example of a newly generated granule cell in the olfactory bulb that is colabeled for
NeuN [arrow, red in (C)] and BrdU [arrow, yellow-green in (D)]. A z-stack series through this cell (56) revealed that the BrdU signal is
confined within and coextensive with the NeuN-labeled nucleus of the cell. Scale bars, 20 µm.
. GFAP or vimentin
immunoreactivity in wild type, GFAP
- / - , or vimentin - / - mice.
Vimentin immunostaining in corpus
callosum reveals astrocytes,
endothelial cells, and ependymal cells
in wild type mice (a) but only
endothelial and ependymal cells in
GFAP- / - mice (c). In the
hippocampus, vimentin-IR is present
in astrocytes of the wild type mice (e),
but is absent in GFAP- / - mice (g).
No difference in GFAP-IR in the
corpus callosum or hippocampus was
observed between wild type and
vimentin - / - mice (b, d, f, and h). The
sections were counterstained with 50-
fold diluted hematoxylin and
erythrosin. The dentate gyrus of the
hippocampus is depicted by an
interrupted line; full arrows,
astrocytes; arrowheads, endothelial
cells; empty arrows, ependymal cells
lining the ventricle. Bar, 40 µm.
Electron micrographs of astrocytes in situ. a,
b, and e, corpus callosum; c, d, and f,
hippocampus, concave aspect of dentate
gyrus. In wild type mice (a and c), the
astrocytic cytoplasm (A) contains distinct
bundles of IFs (asterisks). In GFAP- / - mice
(b and d), the astrocytic cytoplasm is devoid
of IFs. In vimentin - /- mice (e and f), the
astrocytic cytoplasm contains densely packed
IFs (asterisks). Spinal cord, segment C6-7, the
dorsal funiculus (g-j). Loose bundles of IFs in
wild type mice (g) contrast with more
compacted bundles of IFs in vimentin- /-
mice (i). No IFs were detected in GFAP- / - or
GFAP- /- vim - / - mice (h and j). The
micrographs were chosen as representative of
40 or more astrocytic cell bodies examined
for each of the genotypes and anatomical
locations. A, astrocytic cytoplasm; M,
myelinated nerve fiber; N, astrocytic nucleus;
C, capillary; P, cellular processes of the
surrounding neuropile; e, endoplasmic
reticulum; g, Golgi apparatus; x, myelinoid
body; arrows, mitochondria; A1 and A2, two
bordering astrocytic profiles.
Electron micrographs of
transversally or
longitudinally sectioned IF
bundles in astrocytes in vivo.
Astrocytes in vimentin- / -
mice (b and d) contain more
densely packed IFs than in wild
type mice (a and c) with the
distance between adjacent IFs
being substantially reduced.
These examples are taken from
the dorsal funiculus of the
cervical spinal cord; the
astrocytes were more than
50 µm away from the pial
surface. Mi, mitochondrion.
Immunolocalization of nestin or vimentin in wild type, GFAP- /- , vimentin-/- , and GFAP- / - vim - / -
astrocytes in vitro. Nestin immunoreactivity reveals distinct bundles of IFs in wild type or GFAP- / -
astrocytes (a and b) in contrast to vimentin-- / - or GFAP- / - vim- / - astrocytes, which exhibit only a
very weak and diffuse nestin immunoreactivity (c and d). GFAP immunostaining of wild type and
vimentin-/ - astrocytes reveals similar distinctly filamentous staining patterns (e and f). Arrowheads in c
and d depict cell borders. Bar, 10 µm.
. Electron micrographs of cytoplasm of
wild type, GFAP- / -, vimentin- /- , or
GFAP- /- vim - / - astrocytes in vitro.
Characteristic bundles of IFs (asterisks) in
the cytoplasm in wild type astrocytes (a).
IFs are sparse in GFAP- / - astrocytes (b;
inset shows scattered IFs at higher
magnification, thin arrows). In vimentin- /-
astrocytes, IFs are present in the form of
highly contrasted, densely packed bundles
(c; x, dense bundles of IFs; inset shows
details of a bundle of densely packed IFs).
No IFs are present in GFAP- / - vim - / -
astrocytes (d; inset shows detail of the
cytoplasm with microtubules). The
micrographs were chosen as
representative of 30 astrocytic cell bodies
examined for each of the four genotypes.
IFs in a-c are 9-10 nm thick. Mi,
mitochondrion; thick arrows, endoplasmic
reticulum and/or cluster of ribosomes;
arrowheads, microtubules.
Reactive astrocytes in cortical lesions in
GFAP- /- , vimentin- / -, or GFAP- / -vim- / -
mice. Astrocytes in cortical lesions 3 days
following the injury (a-f). Nestin immuno-
staining reveals distinct reactive astrocytes in
wild type or GFAP- / - mice (a and b). In the
corresponding areas in vimentin- / - or
GFAP- / -vim- / - mice, only weak and diffuse
astrocytic profiles are depicted as nestin-
immunoreactive; the situation closely
resembling nestin immunoreactivity in
cultured astrocytes of the same genotype (Fig.
7, c and d) and indicating absence of IFs.
GFAP immunostaining reveals distinct
reactive astrocytes in the injured cortex of
both wild type and vimentin- /- mice (e and f).
Vimentin and nestin immunoreactivity
2 weeks following the injury (g-j). In cortical
lesions in both wild type and GFAP- /- mice,
nestin immunostaining becomes almost
undetectable 2 weeks after the injury (g and
h). While normal vimentin immunostaining
persists at the site of injury in wild type mice
and permits an easy identification of
individual reactive astrocytes (i), in GFAP- / -
mice, vimentin immunostaining is limited to
the central zone of the injury and has a
diffuse character; endothelial cells are clearly
stained but astrocytic profiles cannot be
distinguished. The sections in g and h were
counterstained with 50-fold diluted
hematoxylin and erythrosin. Arrows,
Partnerships of IF proteins in astrocytes; present examples.
Electron micrographs of astrocytes in situ.
a, b, and e, corpus callosum; c, d, and f,
hippocampus, concave aspect of dentate
gyrus. In wild type mice (a and c), the
astrocytic cytoplasm (A) contains distinct
bundles of IFs (asterisks). In GFAP- / -
mice (b and d), the astrocytic cytoplasm is
devoid of IFs. In vimentin - /- mice (e and
f), the astrocytic cytoplasm contains
densely packed IFs (asterisks). Spinal cord,
segment C6-7, the dorsal funiculus (g-j).
Loose bundles of IFs in wild type mice (g)
contrast with more compacted bundles of
IFs in vimentin- /- mice (i). No IFs were
detected in GFAP- / - or GFAP- /- vim - / -
mice (h and j). The micrographs were
chosen as representative of 40 or more
astrocytic cell bodies examined for each of
the genotypes and anatomical locations. A,
astrocytic cytoplasm; M, myelinated nerve
fiber; N, astrocytic nucleus; C, capillary; P,
cellular processes of the surrounding
neuropile; e, endoplasmic reticulum; g,
Golgi apparatus; x, myelinoid body;
arrows, mitochondria; A1 and A2, two
bordering astrocytic profiles.
Figure 3. Pseudocolor laser scanning confocal
micrographs (stereopairs). A, Case 5.
Operculum with numerous cones (original
magnification ×1000) measuring 100 × 130 µm
triple labeled with anti–phosphodiesterase
gamma (PDEG) (red), antirhodopsin (green),
and 4',6'-diamidino-2-phenylindole (DAPI) for
nuclei (blue). A total of 46 cones can be seen
with positive reactivity to anti-PDEG (red) only
and nuclear DAPI (blue). The nuclei (PDEG-
positive and rhodopsin-positive) appear pink,
while cone axons (PDEG-positive) appear red.
Also seen is a population of cells with positive
DAPI nuclear labeling (blue) and negative
labeling to both anti-PDEG and antirhodopsin.
B, Case 6. Operculum (original magnification
×800) measuring 140 × 100 µm with a total of
41 cones. C, Case 10. Operculum (original
magnification ×1000) measuring 95 × 60 µm
triple labeled with anti-PDEG (red), anti–glial
fibrillary acid protein (GFAP) (green), and
DAPI (blue) for nuclei. A total of 14 cones are
seen with positive reactivity to anti-PDEG and
DAPI. Two adjacent glia with typical long
processes are seen with strong reactivity to
anti-GFAP (green).
Neuron Glia cell

Two type of intermediate filaments in cells of nervous system


Neurofilaments

Neurofilaments (NFs) are neuron-specific intermediate filaments, the


predominant cytoskeletal component in large myelinated axons. In
mammals, neurofilaments consist of three protein subunits, known as high
(NF-H, 110KD), middle (NF-M, 90KD) and low (NF-L, 61DK) molecular
weight neurofilament proteins.
Neurofilaments are one of the major components of the neuronal cytoskeleton
and are responsible for maintaining the calibre of axons. They are modified by
post-translational changes that are regulated in complex fashions including by the
interaction with neighbouring glial cells. Neurofilament accumulations are seen
in several neurological diseases and neurofilament mutations have now been
associated with Charcot-Marie-Tooth disease, Parkinson's disease and
amyotrophic lateral sclerosis.
Clustering of neuronal inclusions in "dementia with neurofilament inclusions"

Fig. 1. NI in the superior temporal gyrus in a case of dementia with NI. Section immunostained with phosphorylated SM131 antibody to heavy
weight neurofilaments, counterstained with haematoxylin (NI neurofilament inclusion
Variety of neuronal staining is demonstrated with
several antibodies. Normal NF distribution with
antibody NF-05 is shown in a 79-year-old male (5 h
postmortem delay) without neuropathological disorder
(a). (b,c) Tissue from a 94-year-old female with mild
AD; (d–i) tissue of an 84-year-old female with severe
AD; (j,k) tissue of a 94-year-old female, with mild AD.
(b) and (c) correspond to consecutive sections, which
were stained with antibody NF-03 (b), and with
antibody AT8 (c), a marker for AD-type
phosphorylation of tau. Note that similar structures,
mainly neuropilar threads and neuronal cell bodies,
were stained by both antibodies. (d, f-j) show NF-03
staining, (e) represents NF-04 staining and (a) and (k)
represent NF-05 staining. Bar = 20 µm (a,d,e,g-k) and
40 µm (b,c,f).
Entorhinal cortex of a 84-year-old female with severe AD, (number 8
in Fig. 1c), was used for double-labeling experiments with polyclonal
anti-tau (in red) and monoclonal anti-NF-H, NF-03 (in green).
Secondary antibodies anti-rabbit Ig conjugated to Texas red and anti-
mouse Ig conjugated to Oregon green were used to visualize staining
by confocal microscopy. Note that there is little overlap of tau and NF
staining in form of a yellow color. Cells with a thickness of 16 and 25
µm, respectively, were scanned with a separating distance of 500–800
nm between the focal planes. (a–c) represent the sum of all focal
planes (extended focus), while individual focal planes at an interval of
4 µm and 2 µm, respectively, are shown at the bottom of (b) and (c).
Bars = 50 µm (a), 25 µm (b,c).
Selective Degeneration of Purkinje Cells with Lewy Body-Like Inclusions in Aged
NFHLACZ Transgenic Mice
Pang-hsien Tu1, Kathryn A. Robinson1, Femke de Snoo1, Joel Eyer2, Alan Peterson3,
Virginia M.-Y. Lee1, and John Q. Trojanowski1

NFHLacZ inclusions in neuronal perikarya are


more resistant to trypsin digestion than native
NFs in axons of the corpus callosum in control
mice. A and B illustrate the corpus callosum of a
wild-type (WT) mouse, whereas C and D show
NF inclusions in the neocortex of a NFHLacZ
(TG) mouse. The sections in A and C were
stained with the mAb RMO189 before trypsin
treatment (TS ), whereas the adjacent sections
in B and D were stained with the same mAb
after trypsin treatment (TS+). Although
immunoreactive NFM in normal axons of the
corpus callosum is completely eliminated by
trypsin digestion in the wild-type mouse, the
same does not eliminate NFM immunoreactivity
in the NFHLacZ inclusions of the NFHLacZ
transgenic mouse. The sections in A-D were
counterstained with hematoxylin. A-D are at the
same magnification; scale bar: D, 50 µm.
Quantitative analysis of interactions between tailless neurofilament triplet proteins.
SFY526 cells co-transformed with plasmids derived from pGBT9 (top line) and pGAD424
(bottom line) were grown to mid-log phase in minus Trp-Leu media. Cells were
permeabilized and assayed for -galactosidase activity as described in the text. H , M ,
and L indicate the tailless constructs NFH1-415, NFM1-421, and NFL1-415, respectively. The
backgrounds were measured as the -galactosidase activity, which was obtained from the
co-transformation of pGAD424 and the corresponding pGBT9 NF derivatives and were
subtracted from each set of data.
The influence of the amino-terminal head region
on NFTPs interactions. A, interactions between
partial headless/tailless NFL and tailless NFTPs;
B, interactions between partial headless/tailless
NFM and tailless NFTPs. H , M , M , L ,
and L represent the constructs coding for
NFH1-415, NFM1-421, NFM44-421, NFL1-415, and
NFL24-415, respectively.
. Alignment of human and rat NF-L rod domains. Positions of -helical regions 1A, 1B, 2A, and
2B and linker regions L1, L2, and L12 are indicated. Sequence differences between rat and
human (*) are indicated. The bold symbols represent amino acids that are referred to in the text.
Schematic representation of tomaculi
formation in MAG-deficient
paranodal regions. A-C, Relationship
between axonal caliber and myelin
sheath in transverse section of normal
myelinated fiber (A). In MAG-deficient
nerves, paranodal axonal calibers
shrink (B) and result in myelin sheath
collapse and tomaculi formation (C). D,
In MAG+/+ paranodal regions,
increased neurofilament spacings
correlate with large axonal caliber. In
MAG- / - paranodal regions
surrounded by tomaculi,
neurofilament spacings and axonal
calibers are similar to those in nodal
regions.
Axonal morphology in dorsal and ventral roots
of old NF-null mutant animals. Light
microscopy of toluidine blue–stained cross
sections of L4 dorsal (A, C, E, and G) or L4
ventral (B, D, F, and H) roots from 2-yr-old
wild-type (A and B), NF-M–null mutant (C and
D), NF-H–null mutant (E and F), or NF-M/H–
null mutant (G and H) mice. Insets show cross
sections of whole roots at lower magnification.
The ventral root axons in the NF-M– and NF-
M/H–null mutants appear shrunken and
frequently irregular in shape. In the dorsal roots
only axons in the NF-M/H–null mutant are
affected by the atrophic process. Bar, 10 µm.
Fine structure of axons in old mice with NF-null mutations. Lumbar root axons from 2-yr-old
wild-type (A), NF-M– (B), NF-H– (C), and NF-M/H– (D) null mutant mice are shown. In ventral
roots of the NF-M–null mutant (B), NFs are sparse and MTs are plentiful, whereas axoplasm of
the NF-M/H mutant (D) contains only MTs. Wild-type (A) and NF-H–null mutant (C) show
numerous NFs and fewer MTs. Bar, 200 nm.
Time course of changes in ventral roots
of old NF-null mutant animals. Light
microscopy of toluidine blue–stained
cross sections of L5 ventral roots from
wild-type (A, D, and G), NF-M–null
mutant (B, E, and H), and NF-M/H–null
mutant (C, F, and I) at 4 mo (A–C), 1 yr
(D–F), or 2 yr (G–I) of age. Myelinated
axons in the ventral roots of the 4-mo-
and 1-yr-old NF-M– and NF-M/H–
deficient animals, although reduced in
size, are otherwise normal in
appearance. By contrast, axons in the
ventral roots of the 2-yr-old NF-M– and
NF-M/H–null mutants appear shrunken
and frequently irregular in shape. Bar,
10 µm.
Hind limb paralysis in 2-yr-old NF-M/H–null mutant animal. In A, a 2-yr-old NF-M/H–null
mutant animal is shown which exhibits a hind limb paralysis. Note the abnormal posture of
the hind limbs resulting from an inability to extend the hind limbs in comparison to a wild-
type mouse shown in B.
Figure 1. NF-Mtail does not alter the subunit ratio of
neurofilament subunits. (A) Construction of NF-Mtail results in a loss
of 426 of the 449–amino acid tail of NF-M protein, and this
region is replaced with a Myc epitope tag. The three exons
of NF-M gene are indicated by the black boxes and
interrupted by two introns. ATG denotes the translation
initiation codons. Dotted lines indicate the regions that
underwent homologous recombination between the
targeting vector and the endogenous NF-M allele. (B and
C) Identification of NF-Mtail mice. (B) Genomic DNA blots
of mouse tails hybridized to the region denoted as 5' probe
in A. (C) A similar blot as in B but hybridized with the
sequence in A marked 3' probe. DNA are from littermate
mice with two wild-type NF-M alleles (lane 1),
heterozygous (lane 2), or homozygous (lane 3) for the NF-
Mtail allele. (D–N) NF-M tail deletion does not change NF
subunit ratio but increases axonal tubulin. Sciatic and
optic nerve extracts from wild-type (lanes 1, 4, 7, and 9),
NF-Mtail heterozygous (lanes 2 and 5), and NF-Mtail
homozygous (lanes 3, 6, 8, and 10) mice were fractionated
on 7% SDS–polyacrylamide gels and stained with
Coomassie blue (D) or immunoblotted with antibodies that
recognize (E) NF-M in a phosphoindependent manner; (F)
NF-Mtail with Myc antibodies; (G) NF-L; (H) NF-H; (I)
NF-H phosphospecific, RT-97; (J) NF-H phosphospecific,
SMI-31; (K) NF-H dephosphospecific, SMI-32; (L) -tubulin;
(M) neuron-specific ßIII-tubulin; and (N) Myosin Va. (D) In lanes 11 and 12, quantification standards

at right are provided by a twofold dilution series of a neurofilament preparation. Samples in lanes 1–3

and 7–8 are from 2-mo-old mice, and lanes 4–6 and 9–10 are from 6-mo-old mice. (O) Microtubule

density in motor axons from L5 ventral roots of wild-type or littermate homozygous NF-Mtail

mice. Density was measured by counting all microtubules


in a given cross-sectional area. Counts are an average from
11 axons for each genotype of 6-mo-old animals. Error
bars represent SD.
Figure 2. NF-M tail is important for radial
growth of motor axons. (A) Cross sections of
L5 motor (ventral root) axonal profiles from
wild-type, NF-Mtail heterozygous, and NF-
Mtail homozygous mice at (top) 2 mo or
(bottom) 6 mo of age. Bar, 20 µm. (B and C)
Numbers of small (<4 µm diam; dotted bars)
and large (>4 µm diam; hatched bars) axons
in L5 motor roots of (B) 2- or (C) 6-mo-old
wild-type and NF-Mtail heterozygous and
homozygous mice. Counts are an average
from three to four animals for each genotype.
(D and E) Distributions of axonal diameters
in motor axons of (D) 2- or (E) 6-mo-old
wild-type and homozygous animals. Points
represent the average distribution of axon
diameters form the entire roots of four mice
for each genotype and age group. Error bars
represent SD.
Figure 3. NF-M tail is essential for calibers of
sensory axons. (A) Cross sections of L5 sensory
(dorsal root) axonal profiles from wild-type,
NF-Mtail heterozygous, and NF-Mtail
homozygous mice at (top) 2 or (bottom) 6 mo of
age. Bar, 20 µm. (B and C) Numbers of small
(<4 µm diam; dotted bars) and large (>4 µm
diam; hatched bars) axons in L5 sensory roots
of (B) 2- or (C) 6-mo-old wild-type and NF-Mtail
heterozygous and homozygous mice. Counts
are an average from three to four animals for
each genotype. (D and E) Distributions of
axonal diameters in sensory axons of (D) 2- or
(E) 6-mo-old wild-type and NF-Mtail
homozygous animals. Points represent the
average distribution of axon diameters form
the entire roots of four mice for each genotype
and age group. Error bars represent SD.
Figure 4. Altered neurofilament organization in NF-Mtail axons. Thin-section electron micrographs of 6-mo-old motor axons of the L5 ventral roots of
normal (A) or NF-Mtail
(B) animals. Quick freeze deep etch micrographs of sciatic nerves from (C) wild-type and (D) NF-Mtail
mice were imaged by quick freeze deep etch microscopy. (C' and D') Higher magnification views of areas boxed in C
and D. (long arrows) Fine long cross-bridges; (short arrows) short cross-bridges. The majority of the fine long cross-
bridges are missing in NF-Mtail axons. Bars: (A) 500 nm; (C and C') 200 nm.
Neurofilament-dependent
organization of axoplasm. In normal
axons (A), axoplasm is organized into a
volume-determining three-dimensional array
by a series of linkages that span between
adjacent neurofilaments (blue-gray) and
between neurofilament and microtubules (red)
or cortical actin (blue) filaments. NF-M
(turquoise) and NF-H (purple) tails form cross-
bridges between neurofilaments (Nakagawa et
al., 1995; Chen et al., 2000, Rao et al., 2002b).
Neurofilaments, microtubules, and cortical
actin are interlinked by plectinlike linkers
(orange; Errante et al., 1994; Svitkina et al.,
1996). The neuronal cytoskeleton is also
stabilized by putative cytoskeletal cross-linker
proteins between neurofilaments,
microtubules, actin in motor and sensory axons
(Yang et al., 1996; Dalpe et al., 1998; Leung et
al., 1999), and neurofilament binding motor
protein myosin Va (Rao et al., 2002b). (B) In
the absence of NF-M tails, NF-H, gigaxonin
and other putative linker proteins, and
elevated levels of myosin Va organize a three-
dimensional array with reduced axonal
diameters to support and maintain altered
axonal volume.
Fig. 1. Sequence alignment of NFL clones, location of mutations and
western-blotting of transfected proteins with anti-NFL antibody. (A)
Multiple sequence alignment of the hNFL protein encoded by our hNFL
cDNA (hNFL), the previously published hNFL sequence (hNFL
P09716), and the published rNFL sequence (rNFL). All the differences
in the protein sequences are marked by vertical boxes. Differences in the
amino acid sequence between hNFL and hNFL (P09716) are marked
with an asterisk (*), while amino acids that differ between hNFL and
both hNFL (P09716) and rNFL are indicated with a hash mark (#). (B)
Schematic representation of NFL protein and the positions of the
mutant and variant amino acids. The head, rod and tail domains are
indicated. The rod domain is divided into coils 1A, 1B, 2A and 2B
separated by linkers L1, L12 and L2. The CMT2 mutations are: P8R in
the head domain and Q333P in the coil 2B. The D469N variant is in the
tail domain. (C) Western blots of transfected human (left) and rat
(right) NFL proteins. Cytoskeletal extracts of transfected cells were
separated by SDS-PAGE and subjected to western blotting with a
polyclonal anti-NFL antibody. The antibody recognized a band of the
same size in wild-type NFL (wt), variant NFL (D469N), head mutant
NFL (P8R), and rod mutant NFL (Q333P). The bar indicates the
position of the 64 kDa size marker.
Fig. 2. Co-transfections of wild-type and variant rNFL with
rNFM or rNFH in SW13Vim- cells. (A-D) Rat NFM was co-
transfected with wild-type rNFL (A,B) or D469N variant
rNFL (C,D). The cells were double-labeled with polyclonal
anti-NFL antibody (A,C) and monoclonal anti-NFM antibody
(B,D). For both wild-type and variant NFL, a filamentous
network throughout the cytoplasm was observed that stained
with both antibodies. (E-H) Rat NFH was co-transfected with
wild-type rNFL (E,F) or D469N variant rNFL (G,H). The cells
were double-labeled with polyclonal anti-NFL antibody (E,G)
and monoclonal anti-NFH antibody (F,H). A filamentous
network was formed with both wild-type and variant rNFL
co-transfected with rNFH. In all the subsequent experiments,
the variant NFL behaved identically to wild-type NFL. Bar,
25 µm.
Fig. 3. Co-transfections of mutant rNFL with rNFM or rNFH in SW13 Vim- cells. (A-F) Rat NFM was
co-transfected with P8R mutant rNFL (A-D) and Q333P mutant rNFL (E,F). The cells were double-
labeled with polyclonal anti-NFL antibody (A,C,E) and monoclonal anti-NFM antibody (B,D,F). The
P8R mutant rNFL co-transfected with rNFM either formed a bundle of filaments (A,B) or displayed
punctate or aggregate staining (C,D). The Q333P mutant rNFL resulted in aggregates that contained
rNFM (E,F). (G-L) Rat NFH was co-transfected with P8R mutant rNFL (G-J) and Q333P mutant
rNFL (K,L). The cells were double-labeled with polyclonal anti-NFL antibody (G,I,K) and monoclonal
anti-NFH antibody (H,J,L). The P8R mutant co-assembled into bundled filaments with rNFH in most
cases (G,H), or into more punctate material or aggregates (I,J), whereas the Q333P mutant rNFL
resulted only in the formation of aggregates with rNFH (K,L). Bar, 25 µm.
Fig. 4. Transfection of hNFL clones in SW13
Vim- cells. (A-D) Transient transfection results
of wild-type (A,B), P8R mutant (C) and Q333P
mutant (D) hNFL. Staining was carried out with
monoclonal anti-NFL antibody. Wild-type
hNFL generally assembled into short filaments
(A), but occasionally also formed a
homopolymeric filamentous network (B) that
was not stained with anti-vimentin antibody
(not shown). The P8R and the Q333P hNFL
mutants (C,D) were not capable of self-
assembly into a filamentous network, but
formed aggregates. (E-F) Co-transfections and
staining of wild-type hNFL with the P8R
mutant hNFL (E) and Q333P mutant hNFL (F)
showed that the mutant proteins disrupted the
self-assembly of wild-type hNFL. Results
similar to those in F were obtained with a
bicistronic construct that expressed both wild-
type and Q333P mutant hNFL (not shown).
Bar, 25 µm.
Fig. 5. Co-transfection of hNFL constructs with
hNFM in SW13 Vim- cells. hNFM was co-
transfected with either wild-type hNFL (A,B),
P8R mutant hNFL (C,D) and Q333P mutant
hNFL (E,F). The cells were double-labeled with
polyclonal anti-NFL (A,C,E) and monoclonal
anti-NFM (B,D,F) antibodies. A filamentous
network throughout the cytoplasm was
observed for wild-type hNFL and hNFM (A,B).
The P8R mutant hNFL formed bundled
filaments that stayed on one side of the cell
(C,D; cell on the left) or around the nucleus
(C,D; cell on the right). The Q333P mutant
NFL always formed aggregates with hNFM
(E,F). Bar, 25 µm.
Fig. 6. Transient transfection of hNFL constructs in
SW13 Vim+ cells. Transfection of wild-type (A,B), P8R
mutant (C,D) and Q333P mutant (E-H) hNFL. The cells
were double-labeled with monoclonal anti-vimentin
(A,C,E,G) and polyclonal anti-NFL (B,D,F,H)
antibodies. Both wild-type hNFL (A,B) and D469 hNFL
(not shown) incorporated into the endogenous vimentin
network. The P8R mutant hNFL protein incorporated into
the endogenous vimentin network in most cases (C,D).
The co-assembly of the Q333P mutant hNFL with
endogenous vimentin was dependent on their relative
levels of expression (E-H). At lower levels of expression
the mutant Q333P hNFL incorporated into the vimentin
network (E,F), while at higher levels of expression it
aggregated in the cytoplasm (G,H). Bar, 25 µm.
Fig. 7. Transfection of
hNFL does not affect the
microtubule network of
SW13 Vim- cells.
Transient transfection of
wild-type hNFL (A),
D469N variant hNFL (B),
P8R mutant hNFL (C) and
Q333P mutant hNFL (D)
into SW13 Vim- cells. Cells
were co-stained with
monoclonal anti-ß-tubulin
antibody (green) and
polyclonal anti-NFL
antibody (red). Shown are
the merged images
corresponding to staining
with both antibodies.
Transfection of hNFL
constructs did not affect
the microtubule network,
and NFL and tubulin did
not colocalize.
A) Percentage of cells transfected with single hNFL
constructs displaying each phenotype. N represents the
total number of cells scored for each transfection. The
data were analyzed by using one-way ANOVA followed by
the Bonferroni's Multiple comparison test. The level of
significance was set at P<0.05.

(B) Phenotypes observed in co-transfections of hNFL(WT)


with mutant or variant hNFL cDNA. N represents the
total number of cells scored for each transfection. The
data were analyzed by using the unpaired t-test. The level
of significance was set at P<0.05.

(C) Phenotypes observed in co-transfections of hNFL


cDNAs with wild-type hNFM. N represents the total
number of cells scored for each transfection. Co-
transfection of hNFM with Q333P hNFL mutant always
resulted in the formation of aggregates, while co-
transfection with wild-type of D469N variant hNFL
always resulted in the formation of a filamentous network,
with equal distributions of cells with predominantly thick,
thin or mixed filaments. Co-transfection of hNFM with the
P8R hNFL mutant resulted in an increased number of
cells displaying only thick filaments, while no transfected
cells had only thin filaments.

*
P<0.01 versus WT and D469N.
P<0.001 versus WT and D469N.

P<0.05 versus D469N.

P<0.01 versus WT.

¶ P<0.01 versus D469N.

** P<0.001 versus WT.


NF-L

Co-localization of hamartin and tuberin with NF-L in cultured primary rat cortical neurons. For confocal images of
growth cones, 0.34-µm Z-slices were taken. One slice representing the middle of a growth cone is shown (A-C).
Hamartin, detected with anti-hamartin antibody HF6 and Alexa488-conjugated anti-rabbit antibody, is observed
plainly in the proximal and central regions of the growth cone (A). NF-L is abundant in the same region of growth
cone and in neurite as stained with NF-L-specific antibody and Cy3-conjugated secondary antibody (B and E).
Overlapping images reveal co-localization of hamartin and NF-L in the proximal to central portion of growth cone
(C). Compilation of two slices representing the middle of a growth cone is shown for tuberin and NF-L co-localization
(D-F). Tuberin, detected with C20 and Cy2-conjugated anti-rabbit antibody, reveals localization all along the neurite,
in the growth cone, as well as in filopodial extensions (D, see arrows). NF-L detected as described above is seen in
neurite and proximal to central region of growth cone (E). Overlapping images show co-localization of tuberin and
NF-L (F). Scale bar, A-F, 50 µm.
. A model of the binding partners of hamartin and their relationship to cytoskeletal
components, suggesting hamartin as a novel integrator of the neuronal cytoskeleton.
Hamartin and tuberin associate with each other through their respective N termini.
Hamartin links actin and NF-L filaments through its direct interaction with ERM proteins
and NF-L.
Figure 1. Actin Assemblages(A) Actin-rich projections in the cochlea exhibit defined lengths and
positions.(B) Semicrystalline actin arrays in striated muscle.(C) Zones of actin assembly (arrows)
detected by incorporation of fluorescently labeled actin (red) at the leading edge of a migrating cell.(D)
The actin-rich comet tails (arrows) of Listeria monocytogenes within host cytoplasm.Images shown here
have been reproduced from the following sources: (A) [43], by copyright permission of the Rockefeller
University Press; (B) [37], by copyright permission of Academic Press, Inc.; (C) [5] by copyright
permission of the Company of Biologists, Ltd; (D) [2], courtesy of Tim Mitchison and Julie Theriot.
Figure 3. Similarities between Actin Assembly on the Listeria Surface and at the Cytoplasmic Face of
the Plasma MembraneA comparison of molecular models for actin assembly by Listeria within its
eukaryotic host (top panel) and at the surface of an uninfected mammalian cell (bottom panel). The
Listeria ActA protein appears to stimulate actin assembly by interacting with the Arp2/3 complex
and members of the Ena/VASP family (E/V) that recruit profilin (P). In an uninfected mammalian
cell, for example, Arp2/3 may be localized and activated at particular regions of the cell where actin
assembly is to be favored. One region of the zyxin protein appears to function like ActA to recruit
Ena/VASP and ultimately profilin. Zyxin also binds -actinin, which could facilitate the cross-
linking of newly assembled actin filaments as well as link zyxin directly to cell surface receptors such
as integrins. When active, vav bound to zyxin may locally reduce actin filament capping, thus
enhancing filament elongation.Cell adhesion to extracellular matrix can stimulate vectorial cell
migration, but how is the adhesive event coupled to directional membrane protrusion? The diagram
illustrates a potential mechanism for insuring the spatial coincidence of sites of substratum
recognition by integrin receptors and the actin assembly machinery necessary for cell surface