Received: 24 February 2019 Revised: 26 August 2019 Accepted: 26 August 2019 Uncorrected manuscript published: 28 August 2019 Published

on: 2 October 2019

DOI: 10.1111/tra.12692

ORIGINAL ARTICLE

A novel strategy to visualize vesicle-bound kinesins reveals

the diversity of kinesin-mediated transport

Rui Yang1,2 | Zoe Bostick3 | Alex Garbouchian3 | Julie Luisi1 | Gary Banker1 |
Marvin Bentley3

1
The Jungers Center for Neurosciences
Research, Oregon Health & Science University, Abstract
Portland, Oregon In mammals, 15 to 20 kinesins are thought to mediate vesicle transport. Little is
2
Department of Biochemistry, Duke
known about the identity of vesicles moved by each kinesin or the functional signifi-
University, Durham, North Carolina
3
Department of Biological Sciences and the cance of such diversity. To characterize the transport mediated by different kinesins,
Center for Biotechnology and Interdisciplinary we developed a novel strategy to visualize vesicle-bound kinesins in living cells. We
Sciences, Rensselaer Polytechnic Institute,
Troy, New York applied this method to cultured neurons and systematically determined the localiza-
tion and transport parameters of vesicles labeled by different members of the
Correspondence
Marvin Bentley, Department of Biological Kinesin-1, -2, and -3 families. We observed vesicle labeling with nearly all kinesins.
Sciences and the Center for Biotechnology Only six kinesins bound vesicles that undergo long-range transport in neurons. Of
and Interdisciplinary Sciences, Rensselaer
Polytechnic Institute, 110 8th Street, Troy, NY these, three had an axonal bias (KIF5B, KIF5C and KIF13B), two were unbiased
12180. (KIF1A and KIF1Bβ), and one transported only in dendrites (KIF13A). Overall, the
Email: bentlm3@rpi.edu
trafficking of vesicle-bound kinesins to axons or dendrites did not correspond to their
Funding information motor domain preference, suggesting that on-vesicle regulation is crucial for kinesin
National Institute of Mental Health, Grant/
Award Number: MH066179 targeting. Surprisingly, several kinesins were associated with populations of
somatodendritic vesicles that underwent little long-range transport. This assay should
Peer Review
The peer review history for this article is be broadly applicable for investigating kinesin function in many cell types.
available at https://publons.com/publon/
10.1111/tra.12692/ KEYWORDS
Kinesin, membrane trafficking, motor protein, neuron, polarity, transport, vesicle

1 | I N T RO D UC T I O N large number of kinesin genes raises a fundamental question: Why are

Neurons are highly polarized cells that require proper localization of
membrane proteins for nearly every aspect of their function. These
membrane proteins are synthesized and processed in the rough endo-
plasmic reticulum and the Golgi apparatus, which are located in the
cell body and proximal dendrites. Proteins are then incorporated into
vesicles that are transported to their appropriate destination by
microtubule motor proteins.1,2 Microtubule plus-end-directed trans-
3
port is mediated by kinesins and minus-end directed transport largely
by dynein. Whereas mammalian cells express only a single dynein
4,5
motor domain, the human genome contains 45 kinesins, some 15 to
20 of which are thought to mediate neuronal vesicle transport.6 The
so many motors required to fulfill what appears to be the same basic
function of moving vesicles towards the plus-end of microtubules?
Some of the kinesin diversity may provide redundancy,7-9 but that
alone is unlikely to explain the size of the kinesin family. Instead, the
diversity could allow different kinesins to confer different transport
parameters, targeting vesicles to specific destinations. Individual
kinesins may also be subject to vesicle-dependent differential regula-
tion, allowing them to confer a variety of transport parameters depen-
dent upon the vesicle to which they are bound.
Identifying all of the vesicles transported by a given kinesin is an
important first step towards understanding the functional diversity
within the kinesin family and defining the mechanisms that underlie

© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

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852
the accuracy of vesicle transport. Tracking vesicles labeled with fluo-
10-14
rescently tagged cargo proteins is common practice, but labeling
vesicles by expressing fluorescent kinesins has proven challenging.
Some Kinesin-3 and -4 family members7,12,14-16 have been used to
track neuronal vesicles, but we know of no examples of successful
vesicle labeling with fluorescently tagged Kinesin-1 and -2 family
members. Here we describe technical improvements to enhance vesi-
cle labeling by fluorescent kinesins. Instead of expressing full-length
kinesins, we express only the vesicle-binding tail region of the motor
protein. To further minimize background fluorescence, we record from
transfected cells at early expression times and focus on low-
expressing cells. For Kinesin-1 second, we also employ an expression
control system to minimize the level of unbound cytosolic kinesins17
and use fluorescent probes with high quantum efficiency and low
extinction coefficients.18 Because the expressed kinesin constructs
lack a motor domain, vesicle movement is mediated only by endoge-
nous motors. This report focuses on the role of kinesins in neuronal
transport but the labeling strategy we describe should be widely appli-
cable as the questions raised in this study are equally relevant in all
eukaryotic cells.
We utilized this novel labeling strategy to complete the first sys-
tematic analysis of the localization and transport parameters of vesi-
cles labeled with members of the Kinesin-1, −2, and − 3 families in
cultured hippocampal neurons. Vesicles bound by different kinesins
displayed a variety of distributions and transport parameters, differing
in their velocity, polarity (ie, their relative preference for entering axon
or dendrite), and the directionality of their movements within axons
or dendrites. Six kinesins labeled vesicles undergoing long-range
transport. Of these, three had an axonal bias (KIF5B, KIF5C and
KIF13B), two moved equally in axons and dendrites (KIF1A and
KIF1Bβ), and one was restricted to dendrites (KIF13A). Other kinesins
labeled distinct populations of somatodendritic organelles that seldom
underwent long-range movements, perhaps indicating a function for
these kinesins beyond long-range transport. Notably, the preference
of vesicle-attached kinesins for axons or dendrites often differed from
the transport preference of their motor domains, as defined in motor-
19-21
domain expression assays. This observation suggests that the
identity of the vesicle that a kinesin binds is a crucial determinant of
that kinesin's transport parameters, an idea we refer to as “on-vesicle
regulation”.
2 | RESULTS
2.1 | Novel strategies to enhance vesicle labeling
with fluorescent kinesin tails
An obvious strategy to evaluate a kinesin's transport parameters is to
express a fluorescently tagged version of that kinesin and monitor the
vesicles it labels. With most kinesins, including Kinesin-1 family mem-
bers, this approach does not result in consistent vesicle labeling. For
example, expression of GFP-tagged KIF5B in cultured hippocampal
neurons resulted in diffuse fluorescence without any visible organelle
labeling (Figure 1A) even though this kinesin is known to transport
YANG ET AL.
multiple vesicle populations in axons and dendrites.3,22-24 Kymo-
graphs from the axon and dendrites of a cell expressing KIF5B-GFP
were devoid of transport events (Figure 1A). Kymograph lines with a
positive slope indicate anterograde transport and those with a nega-
tive slope indicate retrograde transport. This convention is followed in
all figures. Expressing members of the Kinesin-3 family sometimes
results in vesicle labeling,7,15 but labeling is often inconsistent. Expres-
sion of full-length GFP-tagged Kinesin-3 family member KIF13B in
cultured neurons resulted in the labeling of a few punctate structures
that underwent transport (Figure 1C). Presumably full-length GFP-
tagged kinesins bind to the vesicles normally transported by these
motors, but overexpression results in a pool of unbound cytosolic
kinesins that obscures vesicle labeling.
To reduce the pool of unbound fluorescently-tagged kinesins we
employed a series of strategies. First, instead of expressing tagged
full-length kinesins we only expressed the vesicle-binding tail domain.
The majority of full-length kinesin molecules are cytoplasmic and pre-
sumably in an autoinhibited state rather than bound to vesicles.25 We
reasoned that expressing constructs lacking the motor domain may
make the tail domain more accessible to bind vesicles. We previously
found that expression of the kinesin C-terminal domain alone is suffi-
cient for vesicle binding7,26 and engineered kinesins that lack the
motor and first predicted coiled-coil that mediates dimerization.20
Expressing this kinesin “tail” construct alone markedly improved label-
ing of all kinesins (Figures 1–5).
For Kinesin-1s, we implemented a transcription control system
previously developed by Arnold and coworkers.17 Here the kinesin
tail was fused to two other domains: (a) A nuclear localization signal
that causes kinesins not bound to vesicles to translocate into the
nucleus; (b) A DNA-binding zinc finger domain that represses tran-
scription of the kinesin expression vector. Kinesin tails that are not
bound to vesicles are targeted to the nucleus where they inhibit
transcription of further kinesin tail mRNA from the plasmid. Nuclear
pores can only accommodate proteins smaller than 90-110 kDa,27 a
limit most full-length kinesins exceed. Kinesin-1 tails are ~400 resi-
dues long, which puts them below the nuclear pore size limit. This
transcription control system improved Kinesin-1 tail labeling, but it
was not effective for other kinesins, presumably due to their larger
sizes.
To further reduce expression levels, we deliberately recorded
cells after short expression times, starting as early as 3.5 hours
after transfection, and focused on low expressing cells which
exhibited maximal labeling of discrete structures with low cyto-
plasmic background. Finally, for some constructs we used HaloTag
visualized with dye JF549 developed by the Lavis lab.18 The quan-
tum yield and extinction coefficient of JF549 are significantly
higher than GFP, resulting in improved labeling of dim organelles,
thus enabling imaging at earlier expression times. Specifically,
Kinesin-1 tails benefitted from using HaloTag dyes. For all other
kinesin tails, GFP and HaloTag gave comparable results. This is
useful for two-color experiments in which with HaloTags can be
visualized together with a GFP-tagged cargo protein (Figure 6).
For kinesins that have known accessory subunits, these were co-
YANG ET AL. 853

F I G U R E 1 Strategies to enhance vesicle labeling with fluorescent kinesins. Representative images of seven DIV hippocampal neurons
expressing full-length GFP-KIF5B (A), HaloTag-KIF5B tail visualized with JF549 (B), full-length GFP-KIF13B (C) and GFP-KIF13B tail (D) for 6 to
8 hours at comparable levels. In (A) and (B), KLC1a was co-expressed. Yellow lines indicate the axons and dendrites from which high magnification
views and kymographs were generated. For clarity, transport events from kymographs were redrawn as solid black lines. Kymograph lines with a
positive slope indicate anterograde transport and those with a negative slope indicate retrograde transport. This convention is followed in all
figures. Scale bars: low magnification = 20 μm; high magnification = 5 μm

expressed to enhance vesicle labeling, as endogenous expression This combination of strategies markedly improved the ability to
levels of vesicle adaptors were not always sufficient to target record transport of structures labeled with exogenous kinesins.
overexpressed kinesin tails to vesicles. Expression of HaloTag-KIF5B revealed extensive vesicle labeling in
854 YANG ET AL.

F I G U R E 2 Kinesin-1-labeled vesicles move bidirectionally in dendrites, but are biased to the axon. Representative images of seven DIV
hippocampal neurons expressing HaloTag-KIF5B tail (A) or HaloTag-KIF5C tail (B), each co-expressed with KLC1a and visualized with JF549.
Yellow lines indicate the axons and dendrites from which high magnification views and kymographs were generated. Kymographs show that both
KIF5B- and KIF5C-labeled vesicles underwent short, bidirectional movement in dendrites and mostly long-range anterograde movements in
axons. Scale bars: low magnification = 20 μm; high magnification = 5 μm. Graphs show quantification of the number of transport events per
100 μm (C), the average velocity of moving vesicles (D) and their average run length (E). KIF5B: 107 events in 17 cells. KIF5C: 386 events in
17 cells. Error bars show the SEM

axons and dendrites. Kymographs from these regions showed numer- range transport in the axon and dendrites that were not present after
ous transport events (Figure 1B). Similarly, expression of fluorescent expression of full-length KIF13B.
KIF13B tail (Figure 1D) enhanced vesicle labeling over full-length This new labeling strategy enabled visualization of vesicles by
GFP-KIF13B (Figure 1C). Tail-labeled structures underwent long- most members of the Kinesin-1, -2 and -3 families. We obtained
YANG ET AL. 855

F I G U R E 3 Kinesin-2-labeled vesicles are largely restricted to the somatodendritic domain and undergo minimal long-ranged transport.
Representative images of seven DIV hippocampal neurons expressing GFP-KIF3B (A) or GFP-KIF3C (B) tail together with KIF3A tail. Yellow lines
indicate the axons and dendrites from which high magnification views and kymographs were generated. Kymographs show that these vesicles did
not undergo long-range transport events. Scale bars: low magnification = 20 μm; high magnification = 5 μm

consistent labeling of structures with 11 of the 15 kinesins we tested.
For simplicity, we use the term “vesicle” to describe these structures
although kinesin labeling alone cannot differentiate between transport
vesicles, RNA granules, neurofilaments, or other structures kinesins
might interact with. Two-color experiments (Figure 6) show that at
least some of the kinesin tail-labeled structures are vesicles that con-
tain cargo proteins en route to the plasma membrane. The principal
properties of different kinesin-labeled vesicles are summarized in
Table 1. Only KIF5A, KIF17, and two members of the Kinesin-4 family,
KIF21A and KIF21B, failed to label vesicles.
2.2 | Kinesin-1 family members preferentially traffic
in axons
Kinesin-1 family members are thought to transport a wide range of
neuronal cargoes. These include vesicle populations that carry pro-
teins destined for the axonal or dendritic plasma membranes23,28,29 as
well as mitochondria,22,30 lysosomes,31 neurofilaments32 and
33
mRNAs. The majority of Kinesin-1 exists as a tetramer with two
heavy chains and two light chains (KLCs) that act as vesicle adap-
tors.34 Figure 2A shows a cell expressing HaloTag-KIF5B tail along
with untagged KLC1. KIF5B-labeled vesicles in the axon and
dendrites. Kymographs show robust vesicle movement in the axon
with many more anterograde than retrograde events. Quantitative
kymograph analysis of transport events showed that there were fewer
transport events in dendrites, and that these events were divided
equally between anterograde and retrograde directions (Figure 2C).
Transport velocities were comparable in axons and dendrites
(Figure 2D). Vesicles moving in dendrites exhibited slightly shorter ret-
rograde than anterograde run lengths, but this difference was not sta-
tistically significant (P > .14) (Figure 2E). Most cells had a notably
higher labeling intensity in the axon than in any of the dendrites
(Figure 2A and Video S1). In low expressing cells the axonal enrich-
ment was not as obvious, but the transport characteristics of moving
vesicles were the same. Co-expression of KLC2 yielded similar results
(data not shown); little or no vesicle labeling was observed without
co-expression of KLC. KIF5C labeling and transport behavior mim-
icked that of KIF5B (Figure 2B-E and Video S1). Labeled vesicles were
similar in size, shape and distribution. The most notable difference
was that KIF5C-labeled about three times as many vesicles as KIF5B
(Figure 2A-C). KIF5C expression is enriched in brain,35 and neurons
may have KIF5C-specific vesicle populations.
Previous analyses of Kinesin-1 motor domains showed that truncated
KIF5B and KIF5C motor domains both have an axonal preference.19,20,36
856 YANG ET AL.

F I G U R E 4 Kinesin-3-labeled vesicles exhibit a diverse range of transport parameters. Representative images of seven DIV hippocampal
neurons expressing GFP-KIF1A tail (A), GFP-KIF1Bβ tail (B), GFP-KIF13A tail (C) and GFP-KIF13B tail (D). Yellow lines indicate the axons and
dendrites from which high magnification views and kymographs were generated. Kymographs show that KIF1A- and KIF1Bβ-labeled vesicles
undergo short, bidirectional movement in dendrites and axons. Vesicles labeled by KIF13A are restricted to dendrites. Vesicles labeled by KIF13B
move bidirectionally in dendrites and have a strong preference for anterograde movement in the axon. Scale bars: low magnification = 20 μm;
high magnification = 5 μm. Graphs show quantification of the number of transport events per 100 μm (C), the average velocity of moving vesicles
(D) and their average run length (E). KIF1A: 430 events in 22 cells. KIF1Bβ: 683 events in 18 cells. KIF13A: 181 events in 290 cells. KIF13B:
649 events in 15 cells. Error bars show the SEM

The labeling and transport parameters of Kinesin-1-labeled vesicles shows
that most vesicles move in the axon (Figure 2A-C), matching the prefer-
ence of the motor domains. This supports the hypothesis that the KIF5B
and KIF5C motor domains inherently confer axon-selective transport in
the absence of on-vesicle regulation.19 Among the vesicles labeled in den-
drites could be the previously proposed Kinesin-1-mediated dendrite-
selective carrriers that transport AMPA receptors.28,37 This observation
may suggest that dendritic transport by Kinesin-1 requires on-vesicle reg-
ulatory mechanisms.28,38
In contrast to KIF5B and KIF5C, we did not observe vesicle label-
ing with Kinesin-1 family member KIF5A. KIF5A may be specialized
for transport of other structures, including mRNA39 and neuro-
filaments.32,40 If these cargoes bind only a small number of kinesin
dimers or use a different kinesin adaptor, they may not be visible over
cytoplasmic background in our assays.
2.3 | Kinesin-2 family members KIF3A/B/C label
organelles that are restricted to the somatodendritic
region
The Kinesin-2 family consists of two KIF3 heterodimers, KIF3AB and
KIF3AC, and the homodimeric KIF17.41,42 Kinesin-2s have been pro-
posed to participate in axonal and dendritic transport,43,44 but their
neuronal cargoes and transport characteristics are not well under-
stood. Figure 3A shows a cell co-expressing GFP-KIF3B tail along with
unlabeled KIF3A tail (Video S2). GFP-KIF3B tail labeled a population
of vesicles that were restricted to the soma and dendrites. Expression
of GFP-KIF3C tail with KIF3A tail gave similar results (Figure 3B and
Video S2). Quantification of fluorescence intensities resulted in a den-
drite to axon ratio of 4.7 ± 0.7 (n = 18) for KIF3B and 6.3 ± 1.0
(n = 18) for KIF3C. Surprisingly, these organelles seldom underwent
long-range transport. The identity of these organelles is unknown, but
YANG ET AL. 857

(A) GFP-KIF1Bα tail

Axon Dendrite Axon Dendrite Axon Dendrite

Axon

Dend

(B) GFP-KIF1C tail

Axon Dendrite Axon Dendrite Axon Dendrite

Dend

Axon

(C) GFP-KIF16B tail

Axon Dendrite Axon Dendrite Axon Dendrite

Dend

Axon

10 μm
30 s

F I G U R E 5 Some Kinesin-3 family members label vesicles that do not undergo long-range transport. Representative images of seven DIV
hippocampal neurons expressing GFP-KIF1Bα tail (A), GFP-KIF1C tail (B) and GFP-KIF16B tail (C). Yellow lines indicate the axons and dendrites
from which kymographs were generated. Kymographs show that vesicles labeled by these kinesins underwent few long-range transport events.
Low magnification scale bar: 20 μm; high magnification scale bar: 5 μm

they are apparently distinct from the dendritic endosomes that bind 2.4 | Kinesin-3 family members display a diverse
KIF16B16 since they could not be labeled with antibodies against range of localization and transport parameters
EEA1 (data not shown). KIF3AB relies on the accessory protein KAP3
The Kinesin-3 family is large, and its members have been implicated in
for vesicle binding in cilia,45,46 and co-expression of exogenous
many different aspects of vesicle transport. KIF1A and KIF1Bβ are the
KAP3B resulted in comparable labeling (data not shown). The associa-
principal motors associated with synaptic vesicles.12 KIF1A also
tion of KIF3 motors with dendritic vesicles is unexpected, since these
moves secretory granules, alphaherpesvirus particles, and some
motors prefer axonal microtubules or are unpolarized in assays utiliz-
20 dendritically polarized vesicles.7,15,52,53 KIF1Bβ moves dendritically
ing constitutively active motor domains.
polarized mRNPs,54 while KIF13A and KIF13B both play a role in
Expression of the other Kinesin-2 family member, GFP-KIF17 tail,
dendrite-selective transport.7
failed to label vesicles although previous results suggest that it is a
GFP-tagged KIF1A tail labeled vesicles in both axons and den-
motor for dendritic cargoes.44,47-51 One possible reason for the lack
drites (Figure 4A,E and Video S3). Kymographs show that these vesi-
of vesicle labeling is that the expression level of endogenous KIF17
cles were transported efficiently and bidirectionally in both axons and
adaptor proteins is not sufficient to target overexpressed KIF17. Co-
expression of the KIF17 adaptor mLin10 47
or identification of other dendrites. KIF1Bβ also labeled vesicles in axons and dendrites

KIF17 vesicle adaptors may enhance labeling in this assay. (Figure 4B,E and Video S3), similar to KIF1A. In some cells, KIF1Bβ
858 YANG ET AL.

F I G U R E 6 Two-color live-cell imaging can identify the cargos moved by different kinesins. (A) Representative high magnification images and
kymographs of cells expressing the indicated kinesin tails and cargoes. Yellow lines indicate transport events in both channels. HaloTags were
visualized with JF549. Cells with KIF5B tails were cotransfected with KLC1. (B) A table indicating the percentage of kinesin-labeled vesicles that
co-transported with various cargo markers. Anterograde axonal transport and bidirectional dendritic transport were analyzed separately

labeling was enriched in the proximal axon, suggesting that this
kinesin may play a role in targeting proteins to the axon initial seg-
ment. Kymographs showed robust transport in both axon and den-
drites. While the transport characteristics of KIF1Bβ vesicles were
similar to those labeled with KIF1A, KIF1Bβ labeled about two to
three times as many vesicles as KIF1A (Figure 4E). Velocities and run
lengths of KIF1A- and KIF1Bβ-labeled vesicles were comparable in
axon and dendrites (Figure 4F,G). The fact that KIF1A is known to
bind vesicles containing the low-density lipoprotein receptor,7 whose
transport is restricted to dendrites, indicates that this motor binds at
least two distinct vesicle populations with different transport
parameters.
KIF13A-labeled vesicles underwent long-range bidirectional trans-
port in soma and dendrites but were not found in the axon beyond
YANG ET AL. 859

TABLE 1 Kinesin localization in cultured hippocampal neurons

Long-range Tail transport Motor domain

Kinesin family Kinesin tail Accessory subunit Vesicle labeling movement Distribution preference preferencea
Kinesin-1 KIF5A KLC1 or KLC2 No Axonal
KIF5B KLC1 or KLC2 Yes Yes Axon and dendrites Axonal Axonal
KIF5C KLC1 or KLC2 Yes Yes Axon and dendrites Axonal Axonal
Kinesin-2 KIF3AB Yes No Somatodendritic Axonal
KIF3AC Yes No Somatodendritic Weakly axonal
KIF17 No Axonal
Kinesin-3 KIF1A Yes Yes Axon and dendrites No preference Non-selective
KIF1Bα No Soma and proximal dendrites Non-selective
KIF1Bβ Yes Yes Axon and dendrites No preference Non-selective
KIF1C Yes No Soma and proximal dendrites
KIF13A Yes Yes Somatodendritic Dendritic Axonal
KIF13B Yes Yes Axon and dendrites Axonal Non-selective
KIF16B Yes No Somatodendritic
Kinesin-4 KIF21A No Axonal
KIF21B No Non-selective
a
Motor domain preferences are based on Huang and Banker study.20

the initial segment (Figure 4C and Video S3). This distribution is remi- in anchoring vesicles and that long-range transport is mediated by
niscent of vesicles labeled with prototypical dendritically polarized another motor, such as dynein.
7,55-58
membrane proteins. We previously implicated KIF13A as a
motor on dendritically polarized vesicles7 and this result supports that
2.5 | Identifying cargoes carried by specific kinesins
hypothesis. Interestingly, the KIF13A motor domain is axon
selective,20 indicating that on-vesicle regulation is crucial for its polar- The identity of the protein content in vesicles that are bound by spe-
ized transport to dendrites. The closely related KIF13B-labeled vesi- cific kinesins is crucial information for understanding the biological

cles that moved in all neurites, but more vesicles entered the axon function of these motors. To test the viability of kinesin tail labeling

than dendrites. In dendrites KIF13B-labeled vesicles moved bidirec-
tionally (Figure 4D,E and Video S3). KIF13B and KIF13A both labeled
vesicles that exhibit high velocities and run lengths (Figure 4F,G).
KIF13B plays a role in axon formation,59 possibly by transporting PIP3
vesicles into the axon,60 and is also involved in dendrite-selective
transport of transferrin receptor and low-density lipoprotein receptor
vesicles.7 The mechanisms of the on-vesicle regulation that allow
KIF13B to mediate such distinctly different transport patterns are
unknown.
Three other Kinesin-3 family members, KIF1Bα-, KIF1C- and
KIF16B-labeled organelles that were restricted to the somatodendritic
domain, but these organelles seldom underwent long-range transport
(Figure 5). KIF1Bα- and KIF1C-labeled small punctate structures in the
cell body and proximal dendrites, although the identity of the labeled
structures was not apparent. KIF16B labeled large, round structures
that extended throughout the dendritic tree, and had previously been
identified as EEA1-positive endosomes.16 These vesicles were largely
stationary; only a small number occasionally underwent movements at
very slow velocities, as seen previously.16 One possible explanation
for the sporadic transport is that kinesins are bound in an inactive
state and that occasional transport events are initiated by temporary
motor activation. A second possibility is that these kinesins play a role
for identifying cargoes, we systematically co-expressed kinesin tails
with candidate fluorescent cargo proteins and imaged their move-
ments (Figure 6). If two proteins move coincidently—in the same
direction and at the same velocity—it strongly suggests that they are
associated with the same vesicles. Figure 6A shows example kymo-
graphs illustrating the movements of two different kinesins (KIF5B
and KIF13A), each co-expressed with the axonal plasma membrane
protein neuron-glia cell adhesion molecule (NgCAM).10,55,61,62 Previ-
ous work19,21,63 and the behavior of KIF5 tails shown here (Figure 2)
support the hypothesis that members of the Kinesin-1 family
mediate the transport of axonally polarized proteins. However, this
kinesin-cargo interaction has never been shown directly. NgCAM-
GFP-labeled vesicles that moved bidirectionally in dendrites, but
preferentially entered the axon, where they underwent mostly antero-
grade transport (Figure 6A). KIF5B-labeled vesicles overlapped with
NgCAM in axon and dendrites, but KIF13A-labeled vesicles did not.
We systematically analyzed the amount of overlap between three
kinesins (KIF5B, KIF13A and KIF1A) and cargo proteins thought to
move in distinct vesicle populations, including transferrin receptor
(a dendritically polarized protein), brain-derived neurotrophic factor
(BDNF; a marker of secretory granules), and NgCAM (Figure 6B).
More than half of KIF5B-labeled vesicles undergoing anterograde axo-
nal transport also carried NgCAM and 30% of dendritic KIF5B-labeled
860 YANG ET AL.

TABLE 2 Quantitative analysis of kinesin vesicle movement

Axon Dendrites

Anterograde events Retrograde events Anterograde events Retrograde events

Velocity Velocity Velocity Velocity

Kinesin N (μm/s) Events/100 μm (μm/s) Events/100 μm (μm/s) Events/100 μm (μm/s) Events/100 μm
KIF5B 17 1.08 ± 0.07 3.11 ± 0.58 1.19 ± 0.22 0.84 ± 0.33 1.24 ± 0.12 0.89 ± 0.22 1.07 ± 0.13 0.68 ± 0.18
KIF5C 17 1.17 ± 0.07 10.61 ± 1.36 1.18 ± 0.08 3.22 ± 0.58 1.17 ± 0.10 2.08 ± 0.35 1.03 ± 0.07 2.00 ± 0.42
KIF1A 22 1.02 ± 0.12 2.84 ± 0.76 0.89 ± 0.07 3.83 ± 0.65 1.17 ± 0.09 3.69 ± 0.42 1.12 ± 0.07 3.48 ± 0.36
KIF1Bβ 18 1.04 ± 0.05 9.42 ± 1.15 1.07 ± 0.06 9.74 ± 0.79 1.19 ± 0.07 7.09 ± 0.65 1.14 ± 0.06 6.05 ± 0.73
KIF13A 20 NA NA NA NA 1.56 ± 0.06 1.89 ± 0.27 1.60 ± 0.13 1.26 ± 0.18
KIF13B 16 1.23 ± 0.07 12.06 ± 1.75 1.62 ± 0.15 3.47 ± 0.55 1.89 ± 0.13 4.48 ± 0.73 1.81 ± 0.10 4.65 ± 0.52

Note: N, number of cells. Numbers are mean ± SEM, Event number, events/50 μm/min. For each motor between 107 and 683 events were analyzed for
each kinesin.

vesicles exhibited co-movement with NgCAM. Vesicles labeled with
KIF5B did not overlap with vesicles labeled with transferrin receptor.
KIF13A did not co-transport with NgCAM or BDNF granules but over
30% of dendritic KIF13A tail-labeled vesicles co-transported with
transferrin receptor (Figure 6B), consistent with previous reports.7
A significant fraction of KIF1A tail-labeled vesicles were also labeled
with BDNF (15% of axonal vesicles, nearly 40% of dendritic vesicles),
also consistent with prior results.15 Together, these results show that
tail labeling is a powerful tool for identifying the cargoes that are
moved by a particular kinesin and confirm that kinesin-vesicle interac-
tions are highly specific.
Expression of kinesin tails could cause a dominant negative effect.
To address this, we expressed GFP-NgCAM together with Halo-
tagged KIF5B, KIF13A or KIF13B tails and measured the transport
parameters of vesicles undergoing anterograde transport in the axon.
The number of transport events observed when labeling vesicles with
fluorescent cargo proteins varies considerably from cell to cell, but
cells expressing KIF5B—and hence subject to any potential dominant
negative effects—showed more NgCAM transport events than those
expressing KIF13s (data not shown). Vesicle velocity may be a more
sensitive measure of dominant negative effects, since a reduction in
the number of motors bound to each vesicle could result in a reduc-
tion in velocity. NgCAM vesicles moved with an average velocity of
1.2 μm/s when KIF13A tail was expressed (n = 68 events from eight
cells), 1.1 μm/s when KIF13B tail was expressed (n = 168 events from
10 cells), and 1.4 μm/s when KIF5B tail was expressed (n = 217 events
from 14 cells). These results show that expression of the KIF5B tail
domain does not inhibit axonal transport of NgCAM vesicles and sup-
ports the hypothesis that short-term, low-level expression of kinesin
tails does not cause a dominant negative effect.
2.6 | Quantitative analysis reveals diverse transport
parameters of different kinesin-labeled vesicles
One of the principal advantages of this labeling strategy is that it
allows for the quantitative analysis of kinesin-labeled vesicle transport
and the systematic comparison of different kinesins within a single cell
type. In the axon, kinesins mediate anterograde movement, and there
were no significant differences in the anterograde velocities of differ-
ent kinesin-labeled vesicles in the axon (Figures 2D and 4D). All aver-
aged slightly more than 1 μm/s, consistent with many earlier
observations. There were, however, significant differences in the den-
dritic velocities of vesicles bound by different kinesins (Table 2).
KIF13A- and KIF13B-labeled vesicles moved with velocities of ~1.6
and ~1.8 μm/s which were significantly higher (P < .03) than all other
kinesins (which averaged between 1.0 and 1.2 μm/s). Notably, the
velocity of KIF13B-labeled vesicles was significantly greater in den-
drites than in the axon. The fact that KIF13A and KIF13B are particu-
larly effective dendritic transporters is consistent with their role in
dendrite-selective transport.7 Run lengths for vesicles labeled with
different kinesins averaged between 5 and 10 μm in axons and
dendrites.
There were pronounced differences in the number of transport
events among vesicles bound by different kinesins, including differ-
ences in the relative balance between anterograde and retrograde
transport and in the relative number of events observed in axons and
dendrites. To determine if the transport of vesicles labeled with some
kinesins exhibited a bias for the anterograde or retrograde direction,
we calculated a directionality index for each kinesin (DI: the number
of anterograde transport events divided by the sum of anterograde
and retrograde events). If the number of anterograde and retrograde
transport events within either axons or dendrites are the same, the DI
is 0.5. Values above 0.5 indicate an anterograde, and values below 0.5
a retrograde bias (Figure 7A,B). In the axon, KIF5B, KIF5C and KIF13B
all exhibited a strong anterograde bias. In contrast, the number of
anterograde and retrograde movements was roughly equal for both
KIF1A and KIF1Bβ. More than 90% of axonal microtubules are ori-
ented with their plus ends away from the cell body.64,65 The retro-
grade movement of kinesin-labeled vesicles implies that kinesins
remain bound during dynein-mediated transport. This observation is
consistent with models in which the direction of vesicle transport is
regulated by activation and deactivation of opposing motors, which
remain bound to vesicles regardless of the direction of their trans-
port.23,29,66 Our findings also suggest that kinesin-dynein interactions
YANG ET AL.
F I G U R E 7 Quantitative analysis of kinesin-labeled vesicles that
undergo long-ranged transport. (A) The directionality of axonal transport
events of vesicles labeled with different Kinesin-1 and Kinesin-3 family
members. (B) The directionality index of dendritic transport events of
vesicles labeled with different kinesins. (C) The polarity index of vesicles
labeled with these same kinesins. Indices were derived from kymograph
analysis of seven to nine DIV hippocampal neurons expressing the
designated kinesin tail labels. No axonal directional index was generated for
vesicles labeled by KIF13A, as those vesicles were restricted to dendrites
861
are differentially regulated for different kinesins because the relative
directional biases of labeled vesicles are quite different. KIF5B, KIF5C
and KIF13B showed a strong anterograde bias in the axon, whereas
KIF1A and KIF1Bβ moved roughly equally in both directions. In den-
drites, no kinesins labeled vesicles with a strong directionality prefer-
ence (Figure 7B). This is consistent with the mixed orientation of
dendritic microtubules, which could enable kinesins to mediate both
anterograde and retrograde transport.64,67,68
There were also important differences among different kinesins in
the number of transport events observed in axons or dendrites. To
capture these differences, we calculated a polarity index (PI) for each
kinesin, which is a measure of the preference for transport from the
cell body into axons or dendrites (Figure 7C). The PI is defined as the
number of anterograde transport events in the axon divided by the
sum of anterograde evens in axons and dendrites. Several kinesins
labeled vesicles that exhibited polarized transport (Figure 7C). Both
Kinesin-1 motors, KIF5B and KIF5C, were biased to the axon
(PI = 0.68 and 0.82, respectively). A third kinesin with an axonal pref-
erence was the Kinesin-3 family member KIF13B (PI = 0.72). Only one
kinesin was strongly polarized to dendrites, the Kinesin-3 family mem-
ber KIF13A (PI = 0.12). KIF1Bβ did not show a strong preference for
either axon or dendrite, indicated by PI = 0.55. The closely related
KIF1A had a slight preference for dendrites (PI = 0.35).
3 | DISCUSSION
Here we describe a combination of methodological improvements
that enable detection of kinesins bound to vesicles in living cells. To
illustrate the application of this new approach, we systematically char-
acterized the localization and transport parameters of the principal
members of the Kinesin-1, -2, and -3 families that are expressed in
cultured hippocampal neurons. Our experiments reveal a wide range
of transport parameters among different kinesins. Only six of these
kinesins exhibited long-range transport. Surprisingly, several other
kinesins labeled vesicles that underwent little long-range transport.
Co-labeling experiments of kinesin tails with cargo proteins show the
viability of this method for identifying the cargoes moved by each
kinesin. Our results also show that the transport parameters exerted
by a particular kinesin are not defined solely by the properties of its
motor domain but can be modified by on-vesicle regulation. The novel
methods described here will be useful in future studies to address the
mechanisms of differential kinesin regulation by interactions with tar-
get vesicles.
3.1 | Labeling vesicles with expressed kinesin tails
To define the role of different kinesins in vesicle transport, we
describe an improved method for visualizing the vesicles moved by a
particular kinesin. The kinesin tail labeling strategy we describe is a
considerable improvement over expressing fluorescently tagged full-
length kinesins. In principle, this labeling method is capable of visualiz-
ing all of the vesicles that are moved by a particular kinesin, as long as
862 YANG ET AL.

TABLE 3 Kinesin tail constructs and accessory proteins used to label vesicles

Construct Construct design Accession number

Halo-KIF5A tail pCb_CAG-HaloTag-LYGAGADLGAGAGAGAGAG-ZincFinger-LPGI-KRAB NM_008447.4
(A)-GGGSGGGSGGGLYI-KIF5A378-1027
Halo-KIF5B tail pCb_CAG-HaloTag-LYGAGADLGAGAGAGAGAG-ZincFinger-LPGI-KRAB NM_008448.3
(A)-GGGSGCGSGGGLYI-KIF5B376-963
Halo-KIF5C tail pCb_CAG-HaloTag-LYGAGADLGAGAGAGAGAG-ZincFinger-LPGI-KRAB NM_001107730
(A)-GGGSGCGSGGGLYKGGGSGGGSGGGP-KIF5C378-955
KIF3A tail FRB-3myc-GGGSGGGSGGGLYK-KIF3A419-702 NM_001300792.1 variant 2
GFP-KIF3B tail GFP-GGGSGGGSGGGLYK-KIF3B391-747 NM_008444.4
411-796
GFP-KIF3C tail GFP-GGGSGGGSGGGLYR-KIF3C NM_008445.2
GFP-KIF17 tail GFP-GGGSGGGSGGGLYKRTGASGGGSGGGSGGGSGGGTSYPYM- NM_010623.4
KIF17400-1038
GFP-KIF1A tail GFP-3myc-YIDITDTNTVPGGPKL-KIF1A395-1698 NM_001294149.1
GFP-KIF1Bα tail GFP-LYKGGSGG-KIF1Bα387-1150 NM_008441.2
387-1770
GFP-KIF1Bβ tail GFP-LYKGGSGG-KIF1Bβ NM_207682.2
GFP-KIF1C tail GFP-GGGSGGGSGGGLYS-KIF1C400-1103 NM_006612.5
361-1749
GFP-KIF13A tail GFP-GGGSGGGSGGGLYKYSDLELKLRILQSTVPRAR-KIF13A NM_010617.2
GFP-KIF13B tail GFP-GGGSGGGSGGGLYKGGGSGGGSGGG-KIF13B442-1843 NM_001081177.1
458-1317
GFP-KIF16B tail GFP-LYKGGGSGGGSGG-KIF16B -RTV NM_024704.4
GFP-KIF5B full-length GFP-TREFGKLAAIRT-KIF5B NM_008448
GFP-KIF13B full-length GFP-VHDAWRSWNR-KIF13B NM_001081177.1
KLC1a FRB-3myc-LYKGGSGG-mmKLC1a NM_008450.2
55
NgCAM-GFP Sampo et al. Z75013
TfR-GFP TfR-GDPPVAT-GFP M11507
BDNF-mRFP BDNF-GGGPRdPPVAT-mRFP NM_001270630.1

there are enough binding sites available on vesicles. We previously
described a “split-kinesin” assay to determine interactions between a
particular kinesin and any labeled vesicle population of interest.7,8,26
That assay is based on altering the transport properties of a given ves-
icle population if and only if it binds an expressed kinesin tail. While
that assay has proved useful in identifying kinesins that transport dif-
ferent vesicles populations, it is essentially binary, that is, it shows
definitively that some labeled vesicles bind a candidate kinesin but is
difficult to know what fraction of labeled vesicles interact with the
kinesin. Thus, that assay provides no information about the transport
properties of the vesicles that bind a specific kinesin. The tail labeling
strategy described here offers a complementary approach that
enables characterization of the transport properties of the vesicles
bound by a given kinesin. Tail labeling can also identify cargo proteins
in two-color experiments and can provide quantitative information
about the percentage of overlap between different cargoes and
kinesins.
Another advantage of the tail-labeling strategy is that it allows the
direct comparison of all relevant kinesins within a single cell type. We
found in neurons that even closely related kinesins can exhibit quite
different transport parameters depending on the vesicles they bind.
While our experiments were focused on neurons, we expect this
method will provide similar information about the role of kinesins in a
broad range of eukaryotic cell types.
As with any assay, this approach has limitations, particularly when
negative results are obtained. One concern is technical: even in neu-
rons with successful labeling, vesicles are usually dim. Modern sCMOS
or EMCCD cameras can detect dim signals, but there may be vesicle
populations that bind so few kinesin molecules that they are not
detectable above background. A second concern is that in some cases,
co-expression of accessory or adaptor proteins may be required for
adequate vesicle labeling. For example, KIF5B and KIF5C only labeled
vesicles when a KLC isoform was also expressed. In addition, we failed
to detect mitochondrial labeling with these kinesins, probably because
the endogenous levels of Trak/Milton adaptor protein22,30 were insuffi-
cient. Less is known about the vesicle-binding interactions for other
kinesins, and identification of other vesicle adaptors may reveal addi-
tional vesicle populations for specific kinesins.
Another concern is that overexpression of kinesin tail constructs
could cause a dominant negative effect by dimerization with endoge-
nous kinesins to form single-headed motors or through competition
with endogenous motors for a limited number of vesicle binding sites.
We sought to avoid these problems by using constructs that lacked
the first coiled-coil which is important for dimerization and by record-
ing at early expression times. Another potential technical problem is
that the specific label, such as GFP, may alter the specific kinesin
motor behavior, yet in other cases be a good reporter. Experiments
with NgCAM and different kinesin tails (discussed above) showed that
YANG ET AL.
at least for Kinesin-1 dominant negative effects are unlikely. We
expressed comparable tail constructs in previous studies and detected
no change in the extent of vesicle transport and localization.7,8,26 Pre-
sumably, endogenous full-length kinesins can still attach to vesicles
and mediate transport in the presence of overexpressed tails.
Finally, labeling a vesicle with a given kinesin does not formally
prove that this kinesin mediates that vesicle's transport. That said, the
different kinesins we expressed labeled vesicle populations with dis-
tinctly different transport parameters, suggesting that tail binding is
specific for a subset of neuronal vesicles.
3.2 | Neuronal kinesins exhibit diverse transport
parameters
Much is known about how kinesin motor domains behave in vitro and
in living cells,21,36,69-71 but relatively little is known about the behavior
of vesicle-bound kinesins. One surprising outcome from this study is
that only six kinesins, all members of the Kinesin-1 and Kinesin-3 fam-
ilies, exhibited long-range transport. Moreover, the transport parame-
ters of the vesicles labeled by these kinesins often did not match the
preference of their motor domains. For example, KIF13A labeled
dendritically polarized vesicles even though its motor domain prefers
axonal microtubules in cell expression assays.20 Even the Kinesin-1
family members, which we show move axonally polarized NgCAM
vesicles, have been implicated in the transport of dendritically polar-
28,38
ized vesicles. Given that cells generate a multitude of vesicles that
need to be delivered to distinct locations, on-vesicle regulation may
be the mechanism that allows for this.
A further surprising result of this study was that several kinesins
labeled vesicles that underwent little long-range transport. These vesi-
cles appear to fall into three distinct categories: (a) small vesicles in
the soma and proximal dendrites that are labeled by KIF1Bα and KIF1C;
(b) large vesicles in soma and proximal dendrites labeled by KIF3AB and
KIF3AC and (c) large endosomal vesicles labeled by KIF16B that are
present in the soma and throughout the dendritic tree. This is surpris-
ing, because the constitutively active motor domains of these kinesins
move efficiently in neurons when expressed alone20 or cross-linked to
peroxisomes.36,72 However, recent experiments from the Verhey lab
found that monomeric kinesin motor domains cause movement when a
sufficient number of them are targeted to peroxisomes.73 Therefore,
the peroxisome assay, while powerful, may not reflect physiological
vesicle transport behavior. This fact supports the hypothesis that
vesicle-bound kinesins can be regulated.
One possibility may be that these kinesins act as tethers that main-
tain organelles at a particular location within the cell. In non-neuronal
74
cells, KIF1C interacts with Rab6 to mediate Golgi tethering. It is pos-
sible that similar mechanisms regulate the tethering function of kinesins
in neurons. Another possibility is that these kinesins are on the vesicles
in an inactive state, awaiting a signal to trigger short-range transport. In
the latter case, there must be regulatory mechanisms that maintain the
inactive state until activation by a particular signal.
We conclude that the transport parameters of most, if not all,
kinesins are governed by on-vesicle regulation. The mechanism by
863
which such regulation is achieved is not known, but several models
have been proposed. One possibility is that multiple motors—dynein,
myosins, or other kinesins—bind vesicles together and that transport
behavior is the sum of their activities,75-79 which may be regulated in
a vesicle-specific way. A second possibility is that adaptor proteins
selectively activate or inhibit kinesins22,23 and that the regulatory pro-
teins that associate with a given kinesin are vesicle-specific.
A crucial next step towards understanding on-vesicle regulation will
be the identification of the regulatory proteins that interact with vesicle-
bound kinesins. The ability to visualize vesicle-associated kinesins directly
is an important tool towards this goal. For example, two-color experi-
ments with a kinesin and a candidate regulatory protein can confirm their
localization on the same vesicles. Manipulation of candidate regulatory
proteins followed by assessment of changes in the transport parameters
of relevant kinesin-labeled vesicle populations may provide insight into
the regulation of specific kinesins. Because of the complexity of kinesin-
mediated transport and its regulation, we expect labeling of vesicle-bound
kinesins to be a useful tool in many future applications.
4 | MATERIALS AND METHODS
4.1 | Cell culture
Primary hippocampal neurons were prepared as described previ-
ously.80-82 Hippocampi were dissected from E18 rats, trypsinized, dis-
sociated, and plated on poly-L-lysine-treated 18 mm glass coverslips.
Cultures were grown in minimal essential medium with N2 supple-
ments and maintained at 37
C in an incubator with a 5% CO2 atmo-
sphere. Constructs were transfected into stage 4 hippocampal
neurons (8-12 days in culture) with Lipofectamine 2000 (Thermo
Fisher) and allowed to express for 5-18 hours before imaging. For sev-
eral difficult-to-image kinesins it was necessary to focus on the lowest
expressing cells, which was accomplished by recording as early as
5 hours after transfection. This was done to minimize the background
fluorescence caused by soluble kinesins.
4.2 | DNA constructs
Kinesin tail constructs were generated by replacing the N-terminal motor
domain and the dimerization domain with a fluorescent tag. A full list of
kinesin tails used in this study and their correlated amino acid residue
sequence is in Table 3. For Kinesin-1 family members, a zinc-finger DNA-
binding domain and a KRAB(A) nuclear localization and transcription
repressor domain17 were fused to the N-terminus of kinesin tails. The
zinc-finger domain (CCR5ZFL) that recognizes the CCR5 gene83 was
followed by the KRAB(A) domain from rat Kid-1. The DNA recognition
site of CCR5ZFL (50 -gtcatcctcatc-30 ) was inserted upstream of the CAG
promoter to allow for self-regulation by CCR5ZFL/KRAB(A).
Each kinesin construct was designed to exclude the motor and dimer-
ization domains as we had previously established that the tail domain
alone is sufficient for vesicle binding.7,8,26 Every part of the molecule after
the first coiled-coil domain (as defined by the coils predictive algorithm84)
was fused to an N-terminal fluorescent probe. Removal of the first coiled-
864
coil is intended to minimize dimerization with endogenous kinesins, which
could otherwise result in a dominant negative effect. A heterodimer con-
sisting of a full-length endogenous kinesin and a motorless tail would not
be able to processively walk along microtubules.
4.3 | Imaging
For live imaging, cells were maintained in Hibernate E medium without
phenol red (Brain-Bits). Images were acquired using a microscope (model
Ti-E; Nikon) equipped with a spinning-disk confocal-head (model CSU-
W1, Yokogawa), and images were captured with an sCMOS camera (Zyla,
Andor). KIF3AB and KIF3AC images were acquired with an Andor Drag-
onfly built on a Ti2 (Nikon) with a CFI Apo 60× 1.49 objective (Nikon)
and captured with an sCMOS camera (Zyla 4.2+, Andor). The entire imag-
ing stage and objectives were maintained at 37
C in a warmed enclosure
(full lexan incubation ensemble; OkoLab). A Plan-Apo 100× 1.49 NA
objective (Nikon) was used with 2 × 2 binning to acquire image streams.
During image acquisition, z-axis movement was controlled by the Perfect
Focus system on the Ti-E microscope (Nikon). Recordings were acquired
at two frames per second. For further details, see Kaech et al.82
Axons were identified with anti-neurofascin antibody (NeuroMab,
Cat #: 75-027) conjugated to CF405 (Mix-n-Stain CF405S Antibody
Labeling Kit; Biotum, Cat#: 92231) in the imaging medium. Cells
expressing constructs with HaloTag were treated with 50 nM Janelia
Farm 549 dye18 for 10 minutes and washed with conditioned medium
for 10 minutes prior to imaging.
4.4 | Image analysis
MetaMorph software (Molecular Devices) was used to generate kymo-
graphs from movies. Transport events were identified on the kymographs
and the coordinates were exported to Excel (Microsoft) for analysis. It can
be difficult to identify dim transport events with complete certainty. To
ensure consistency, a single reviewer identified transport events on all
kymographs and scored only clear and “indisputable” transport events,
avoiding those that were borderline. Reviewer was blinded to the kinesin
expressed in a given cell and kymographs from all conditions were ran-
domly mixed together to prevent observer bias.
To ensure only microtubule-based long-range transport events
were included in the analysis, excursions <3 μm were excluded. Each
contiguous line with a constant slope was scored as a single transport
event and its velocity and run length (distance moved along the long
axis of the neurite) were calculated. A single vesicle could undergo
multiple transport events if there was a distinct pause between them.
Between 17 and 22 cells were evaluated for each condition, including
cells from at least two independent cultures. For KIF13A tail-labeling
we excluded the first 50 μm of axon from the transport analysis, as
this region has dendritic characteristics and stains positively for the
dendritic marker MAP2.
KIF3AB and KIF3AC dendrite to axon ratios were calculated from
the first frame of each cell recoding. After a background subtraction,
the average intensity of one-pixel wide line scans was used to calcu-
late the ratio. Linescans were at least 10 μm long and measured on the
YANG ET AL.
primary dendrite and the axon, distal to the initial segment. P-values
mentioned in the text were determined by Student's t tests.
ACKNOWLEDG MENTS
We thank Dr Stefanie Kaech for her advice on imaging and Swagatha
Dey for her help in generating the KIF3 constructs. We also thank Drs
Susan Gilbert, Catherine Drerup, Ines Hahn, and André Völzmann and
members of the Bentley lab for their comments on the manuscript.
We thank Geraldine Quinones for excellent technical assistance. We
thank the BioResearch facility at Rensseler for help with tissue collec-
tion for neuronal cultures. This work was supported by National Insti-
tutes of Health grant MH066179.
CONFLIC T OF INT ER E ST
The authors declare no competing financial interests.
OR CID
Marvin Bentley https://orcid.org/0000-0002-1754-0332
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SUPPORTING INF ORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Yang R, Bostick Z, Garbouchian A,
Luisi J, Banker G, Bentley M. A novel strategy to visualize
vesicle-bound kinesins reveals the diversity of kinesin-
mediated transport. Traffic. 2019;20:851–866. https://doi.
org/10.1111/tra.12692