Molecular Biology of the Cell
Vol. 18, 1839–1849, May 2007
Kif5B and Kifc1 Interact and Are Required for Motility
and Fission of Early Endocytic Vesicles in Mouse Liver□
Sangeeta Nath,*†Eustratios Bananis,*†Souvik Sarkar,*†Richard J. Stockert,*
Ann O. Sperry,‡John W. Murray,*†and Allan W. Wolkoff*†
†Department of Anatomy and Structural Biology and *Marion Bessin Liver Research Center, Albert Einstein
College of Medicine, Bronx, NY 10461; and‡Department of Anatomy and Cell Biology, Brody School of
Medicine at East Carolina University, Greenville, NC 27858
Submitted June 19, 2006; Revised February 23, 2007; Accepted March 1, 2007
Monitoring Editor: Erika Holzbaur
Early endocytic vesicles loaded with Texas Red asialoorosomucoid were prepared from mouse liver. These vesicles bound
to microtubules in vitro, and upon ATP addition, they moved bidirectionally, frequently undergoing fission into two
daughter vesicles. There was no effect of vanadate (inhibitor of dynein) on motility, whereas 5?-adenylylimido-diphos-
phate (kinesin inhibitor) was highly inhibitory. Studies with specific antibodies confirmed that dynein was not associated
with these vesicles and that Kif5B and the minus-end kinesin Kifc1 mediated their plus- and minus-end motility,
respectively. More than 90% of vesicles associated with Kifc1 also contained Kif5B, and inhibition of Kifc1 with antibody
resulted in enhancement of plus-end–directed motility. There was reduced vesicle fission when either Kifc1 or Kif5B
activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required
for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody after expression of FLAG-Kifc1 in 293T cells
indicates that these two motors can interact with each other. Whether they interact directly or through a complex of
potential regulatory proteins will need to be clarified in future studies. However, the present study shows that coordinated
activity of these kinesins is essential for motility and processing of early endocytic vesicles.
Receptor-mediated endocytosis is a process in which ligands
bind to specific cell surface receptors and internalize via
clathrin-coated pits. After internalization the clathrin coat is
released and uncoated vesicles mature into early endosomes
(Mellman, 1996; Mukherjee et al., 1997; Marsh and McMahon,
1999; Higgins and McMahon, 2002; Perrais and Merrifield,
2005). After acidification of early endosomes, ligands such as
asialoorosomucoid (ASOR) that are destined for lysosomes
dissociate from their receptors (Harford et al., 1983a,b), and
a series of fission events results in their segregation into
separate daughter vesicles (Wolkoff et al., 1984; Mellman,
1996; Mukherjee et al., 1997; Murray and Wolkoff, 2003). The
resulting ligand-enriched late endosomes traffic to lyso-
somes for degradation, whereas the receptor-containing
daughter endosomes traffic back to the cell surface where
receptor is reused (Harford et al., 1983a; Wolkoff et al., 1984;
Mukherjee et al., 1997). Previous studies indicated that this
segregation event requires an intact microtubule cytoskele-
ton (Goltz et al., 1992; Novikoff et al., 1996; Murray et al.,
2000; Bananis et al., 2003, 2004). In more recent studies,
microtubule-based endosome motility and segregation were
reconstituted in vitro using fluorescent early endosomes
prepared from rat liver 5 min after portal venous injection of
Texas Red-labeled ASOR, a substrate for the hepatocyte-
specific asialoglycoprotein receptor (ASGPR) (Bananis et al.,
2000, 2003, 2004; Murray et al., 2000; Murray and Wolkoff,
2003). On addition of ATP to the in vitro assay, these vesicles
moved bidirectionally along microtubules.
This in vitro system has permitted identification of native
endogenous proteins that are required for microtubule-
based processing of endocytic vesicles. Using these tools, we
found that plus-end motility of early endocytic vesicles from
rat liver was mediated by the conventional kinesin Kif5B
(standardized nomenclature kinesin-1) (Miki et al., 2003,
2005; Lawrence et al., 2004), and minus-end motility was
mediated by Kifc2 (standardized nomenclature kinesin-14B)
(Bananis et al., 2000, 2003, 2004; Murray et al., 2000). Whereas
Kifc2 was initially described as a brain-specific minus-end–
directed kinesin (Hanlon et al., 1997; Saito et al., 1997), our
studies showed that it was highly associated with these
vesicles prepared from rat liver (Bananis et al., 2003). Al-
though the rat is a convenient experimental animal, the
power of genetic models that have been established in the
mouse led us, in the present study, to examine microtubule-
based motility and processing of early endocytic vesicles
derived from mouse liver. Although, based on rat studies,
we expected to see altered endocytic processing of ASOR by
Kifc2 knockout mice (Yang et al., 2001a), we found no dif-
ferences as compared with wild-type mice. This provided
the rationale to define motors that mediate early endocytic
vesicle motility in mouse liver and to investigate interactions
of these vesicle-associated motors with each other. Such
interactions may be of great importance in coordinating
This article was published online ahead of print in MBC in Press
on March 14, 2007.
VThe online version of this article contains supplemental material
at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Allan W. Wolkoff (wolkoff@aecom.
Abbreviations used: AMP-PNP, 5?-adenylylimido-diphosphate;
ASGPR, asialoglycoprotein receptor; ASOR, asialoorosomucoid.
© 2007 by The American Society for Cell Biology1839
activities of opposing motors to regulate endosome fission
events as well as trafficking of vesicles to specific destina-
tions within the cell.
MATERIALS AND METHODS
Chemicals and Reagents
ASOR was prepared from human orosomucoid (Sigma-Aldrich, St. Louis,
MO) by acid hydrolysis (Stockert et al., 1980) and labeled with Texas Red
(Murray et al., 2000) or125I (Wolkoff et al., 1984) as described previously.
Rabbit polyclonal antibody to the 17 N-terminal amino acids of the H1
subunit of the human ASGPR, which also cross-reacts with mouse ASGPR
(Treichel et al., 1994), and rabbit polyclonal antibody against Kifc1 (Zhang and
Sperry, 2004) were prepared as described previously. Rabbit polyclonal anti-
body against Kifc3 was purchased from Protein Tech Group (Chicago, IL).
Mouse monoclonal immunoglobulin G (IgG) against dynein intermediate
chain (IC) was purchased from Chemicon International (Temecula, CA).
Tubulin was purchased from Cytoskeleton (Denver, CO). Rabbit antiserum
against kinesin-1 (Kif5B) was provided by Dr. Lawrence S.B. Goldstein (Uni-
versity of California, San Diego, La Jolla, CA). Monoclonal antibody against
kinesin-1 heavy chain (H2) was generously provided by Dr. Scott Brady
(University of Illinois, Chicago, IL). Kinesin light chain (KLC) antibody was
purchased from Chemicon International. Cy2- and Cy5-labeled secondary
antibodies were purchased from Jackson ImmunoResearch Laboratories
(West Grove, PA) and Alexa 488-labeled secondary antibody was purchased
from Molecular Probes (Eugene, OR).125Iodine was purchased from GE
Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). All other
reagents were from Sigma-Aldrich unless otherwise stated.
Wild-type C57BL/6J mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). Kifc2 knockout mice were kindly provided by Dr. Lawrence S.B.
Goldstein. Male Sprague-Dawley rats (200–250 g) were purchased from Tac-
onic Farms (Germantown, NY). All animal procedures were approved by the
Animal Institute Committee of the Albert Einstein College of Medicine
Immunoblots were performed as we have described previously (Bananis et al.,
2004). In brief, protein samples were subjected to SDS-polyacrylamide gel
electrophoresis (PAGE) under reducing conditions (100 mM dithiothreitol
[DTT]) and transferred to a polyvinylidene difluoride membrane (PerkinElmer
Life and Analytical Sciences, Boston, MA). The membrane was blocked with
Tris-buffered saline (TBS) (50 mM Tris-HCl and 150 mM NaCl, pH 7.6)
containing 0.1% Tween 20 and 10% nonfat dried milk before incubation with
primary antibody diluted appropriately in TBS, 0.1% Tween 20, and 2%
nonfat dried milk.
Preparation of FLAG-Kifc1 and FLAG-Kifc2 Expression
Kifc1 cDNA (Zhang and Sperry, 2004) was cloned into the pFLAG-CMV-5c
vector (Sigma-Aldrich) by using BamHI and HindIII restriction sites. Kifc2
cDNA (kindly provided by Dr. Lawrence S.B. Goldstein) was cloned into the
pFLAG-CMV-5a vector (Sigma-Aldrich) by using HindIII and EcoRV restric-
tion sites. Transient transfection of 293T cells with these plasmids was per-
formed using PolyFect transfection reagent (QIAGEN, Valencia, CA) accord-
ing to the manufacturer’s instructions. Cells were harvested 2 d after
transfection, washed with phosphate-buffered saline (PBS), and lysates used
for further studies as described below.
Preparation of Brain Lysate
Mouse brain was homogenized in radioimmunoprecipitation assay buffer (50
mM Tris-Cl, pH 8.0, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 150
mM NaCl) containing 1:50 protease inhibitor (catalog no. P-8340; Sigma-
Aldrich). The homogenate was then subjected to centrifugation at 30,000 rpm
for 30 min in an SW60 rotor at 4°C. The supernatant was collected and stored
at ?80°C until use.
Preparation of Antibody to Kifc2
A peptide (GTPSSLSTDTPLTGTSC) containing 17 amino acids near the car-
boxy terminus (amino acids 746–762) of Kifc2 was synthesized by The Lab-
oratory for Macromolecular Analysis and Proteomics at the Albert Einstein
College of Medicine. This peptide was linked to maleimide activated keyhole
limpet hemocyanin (Pierce Chemical, Rockford, IL) according to the manu-
facturer’s directions, and this was used for immunization of rabbits by Co-
vance Research Products (Danver, PA). Crude antiserum was purified on
SulfoLink gel (Pierce Chemical) to which the peptide was coupled according
to the manufacturer’s protocol. Specificity of the antibody was assessed by
immunoblot of liver endocytic vesicles, brain lysate, and expressed FLAG-
Kifc2 (see below) in the presence and absence of peptide.
Isolation and Culture of Mouse Hepatocytes
Hepatocytes were isolated from mice after perfusion of the liver with colla-
genase type 1 (Worthington Biochemicals, Lakewood, NJ) (Xu et al., 1998).
Viability of cells was ?90% as judged by trypan blue exclusion. For studies of
125I-ASOR processing, 1.5 ? 106cells in Waymouth’s 752/1 medium contain-
ing 25 mM HEPES, pH 7.2, 5% heat-inactivated fetal bovine serum, 26 mM
NaHCO3, 5 ?g/ml bovine insulin, 100 IU/ml penicillin, and 0.1 mg of
streptomycin were plated in each 60-mm culture dish (Primaria; BD Bio-
sciences, Franklin Lakes, NJ). The medium was changed after 2 h, and the
cells were maintained in culture for 16–18 h in a 5% CO2atmosphere at 37°C
(Wolkoff et al., 1984). For immunofluorescence studies, cells were cultured on
MatTek culture dishes (MatTek, Ashland, MA) coated with 0.5 mg/ml Matrigel
(BD Biosciences) using hepatozyme medium (Invitrogen, Carlsbad, CA).
Studies of125I-ASOR Internalization and Degradation by
Overnight Cultured Mouse Hepatocytes
Surface binding, internalization, and degradation of125I-ASOR by cultured
hepatocytes were assayed as described in previous studies (Wolkoff et al.,
1984; Samuelson et al., 1988). In brief, 1 ?g/ml125I-ASOR (6700 cpm/ng) was
added to hepatocyte monolayers in ice-cold binding medium (135 mM NaCl,
0.81 mM MgSO4, 1.2 mM MgCl2, 27.8 mM glucose, 2.5 mM CaCl2, and 25 mM
HEPES, pH 7.2) and incubated for 60 min at 4°C. Cells were washed four
times with ice-cold binding medium to remove unbound ASOR, and then the
surface-labeled cells were incubated at 37°C for 0–90 min. Ligand degrada-
tion was quantified as radioactivity remaining soluble after addition of an
equal volume of 20% trichloroacetic acid, 4% phosphotungstic acid to the
incubation medium. Cells were washed twice and surface binding was quan-
tified as radioactivity released following incubation in 20 mM EGTA in 0.15 M
NaCl, 0.02 M Tris-Cl, pH 7.6. Nonspecific binding was assessed by inclusion
of 100 ?g of unlabeled ASOR in the initial incubation with125I-ASOR.
Immunofluorescence Studies in Overnight Cultured Mouse
Cultured cells were exposed to 10 ?g/ml Texas Red ASOR for 5 min at 37°C
in hepatozyme medium, washed twice with warm PBS, and snap-frozen at
?80°C with a minimum of PBS to cover the cells. For immunostaining, cells
were thawed rapidly and fixed at room temperature for 15 min in 4%
formaldehyde at pH 7.4 in 0.25 M sucrose, 5 mM MgCl2, 5 mM EGTA, and 35
mM 1,4-piperazinediethanesulfonic acid (PIPES). They were washed with
PBS containing 5 mg/ml casein and incubated for 40 min in Kifc1 antibody
diluted 1:100 in this solution. After six washes with PBS containing 5 mg/ml
casein, cells were incubated for 40 min in Alexa 488-labeled secondary anti-
body diluted 1:1000. They were washed extensively and observed by fluores-
cence microscopy as described below.
Endosome Isolation and In Vitro Motility Assay
Texas Red-labeled early endocytic vesicles were prepared from mouse or rat
liver as described previously (Bananis et al., 2000, 2003; Murray et al., 2000). In
brief, livers were harvested 5 min after portal venous injection of 50 ?g of
Texas Red-labeled ASOR. A postnuclear supernatant was prepared after
Dounce homogenization of the liver and subjected to chromatography on a
Sephacryl S200 (GE Healthcare) column. Vesicle-enriched fractions were
pooled and centrifuged at 200,000 ? g for 135 min on a sucrose step gradient
consisting of 1.4, 1.2, and 0.25 M sucrose in a Beckman SW60 rotor. Vesicles
were collected from the 1.2 M/0.25 M sucrose interface and stored at ?80°C
until used. Motility assays were performed in a 3-?l chamber consisting of
two pieces of double-sided tape sandwiched between optical glass as de-
scribed previously (Murray et al., 2002). The chamber was coated with 0.03
mg/ml DEAE-dextran (GE Healthcare), and rhodamine-labeled, Taxol-stabi-
lized microtubules were added and incubated for 3 min at room temperature
(Bananis et al., 2000, 2003, 2004). The chamber was washed three times with
PMEE motility buffer (35 mM PIPES-K2, 5 mM MgCl2, 1 mM EGTA, 0.5 mM
EDTA, 4 mM DTT, 20 ?M Taxol, and 2 mg/ml BSA) containing 5 mg/ml
casein followed by three washes with PMEE motility buffer without casein.
Vesicles were then flowed into the chamber, incubated for 10 min to permit
binding to microtubules, and washed with PMEE motility buffer containing
10 mM ascorbic acid. Motility was initiated by the addition of 50 ?M ATP
without a regenerating system in the presence or absence of 5 ?M vanadate
or 1 mM 5?-adenylylimido-diphosphate (AMP-PNP). In some studies to quan-
tify directional motility, polarity-marked, rhodamine-labeled microtubules
were used. These polarity marked microtubules were prepared by first poly-
merizing seeds containing 10 mg/ml tubulin (1:75 labeled/unlabeled) in
polymerizing buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP,
and 3% glycerol, pH 6.8) at 37°C for 5 min. The seeds were sheared by
pipetting up and down and subjected to extension at their ends after addition
of 2.5 mg/ml tubulin (1:6 labeled/unlabeled) for 6 min. The reaction was
stopped by adding polymerizing buffer containing 20 ?M Taxol. Microtu-
bules were pelleted by centrifugation for 4 min at 15 psi at room temperature
S. Nath et al.
Molecular Biology of the Cell1840
in a Beckman airfuge and resuspended in polymerizing buffer containing 20
?M Taxol. In some experiments microtubule-bound vesicles were incubated
with antibodies against specific motor proteins for 6 min before the addition
of ATP (Bananis et al., 2003). In some immunofluorescence colocalization
studies, chambers were not coated with microtubules, and vesicles were
bound directly to the glass surface as we described in previous studies
(Bananis et al., 2000).
Immunofluorescence Studies of Vesicles In Vitro
Glass- or microtubule-bound endocytic vesicles were incubated with appro-
priately diluted primary antibodies in PMEE buffer (35 mM PIPES-K2, 5 mM
MgCl2, 1 mM EGTA, 0.5 mM EDTA, 20 ?M Taxol, and 2 mg/ml BSA)
containing 5 mg/ml casein for 6 min at room temperature, blocked with
motility buffer containing 5 mg/ml casein, and incubated with fluorescently
labeled affinity-purified secondary antibody for 5 min. For simultaneous
immunolocalization of two proteins, vesicles were first washed with motility
buffer containing 5 mg/ml casein after sequential incubation with the first set
of primary and fluorescent secondary antibodies. This was followed by incu-
bations with the second set of primary and contrasting fluorescent secondary
antibodies. The chambers were washed with PMEE motility buffer containing
10 mM ascorbic acid and examined by immunofluorescence microscopy
(Bananis et al., 2000, 2003, 2004).
Imaging was performed with a 60? 1.4 numerical aperture Olympus objec-
tive on an Olympus 1 ? 71 inverted microscope maintained either at 28°C for
mouse vesicle studies or at 37°C for rat vesicle studies and containing auto-
mated excitation and emission filter wheels. Data were collected through a
CoolSNAP HQ cooled charge-coupled device (Photometrics, Roper Scientific,
Tucson, AZ) camera regulated by MetaMorph (Molecular Devices, Sunny-
vale, CA) software. Fluorescent images were analyzed using ImageJ (National
Institutes of Health public domain; http://rsb.info.nih.gov/ij/) and Adobe
Photoshop version 6.0 (Adobe Systems, San Jose, CA), and colocalizations
were scored manually. Colocalization of ASOR-containing vesicles with an-
tibodies to candidate proteins was quantified by first determining the number
of fluorescent vesicles in the rhodamine (ASOR) channel and then overlaying
the green channel to find the number of colocalized vesicles. For motility
studies, time-lapse movies were taken at 1 frame per second for 60 s. Movies
were analyzed using ImageJ software.
Interaction of Kif5B and FLAG-Kifc1
Preliminary studies showed that 293T cells have endogenous expression of
Kif5B (kinesin-1). 293T cells transiently transfected with pFLAG-Kifc1 or
pFLAG vector alone, as described above, were incubated for 60 min on ice in
immunoprecipitation buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50 mM
KCl, 10 mM EDTA, 10 mM EGTA, 1.5% Triton X100, and 0.75% NP-40)
containing protease inhibitors (catalog no. P-8340; Sigma-Aldrich). The lysate
was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was
incubated with nonimmune IgG-linked agarose (Sigma-Aldrich) for 3 h fol-
lowed by overnight incubation with anti-FLAG M2-linked agarose (Sigma-
Aldrich) according to the manufacturer’s instructions. Immunoabsorbed pro-
tein was identified by immunoblot. In companion studies, 293T cells were
cotransfected with FLAG-Kifc1 and a pcDNA3 expression plasmid encoding
KLC2 (Ligon et al., 2004), kindly provided by Dr. Erika Holzbaur (University
of Pennsylvania). These cotransfected cells were processed as described above
to assess interaction of Kifc1 with KLC2.
Statistical analysis was performed using chi-square or Student’s t test as
Binding, Internalization, and Degradation of125I-ASOR
by Wild-Type and Kifc2 Knockout Mouse Hepatocytes
Based upon previous studies in rat early endocytic vesicles
showing that Kifc2 mediated their minus-end–directed mo-
tility on microtubules (Bananis et al., 2003), we hypothesized
that Kifc2 knockout mice would show a defect in endocytic
processing of ASOR. This was examined by quantifying
single-wave processing of125I-ASOR by overnight cultures
of hepatocytes isolated from livers of wild-type and knock-
out mice. In cells from both types of mouse, internalization
of cell surface bound125I-ASOR was rapid with ?70% in-
ternalized within 30 min of incubation at 37°C (Figure 1A).
Degradation of internalized ligand was nearly complete by
90 min in both wild-type and knockout cells (Figure 1B),
indicating that internalization and processing of125I-ASOR
in mouse liver do not require Kifc2, but likely use another
microtubule-based motor(s). This was examined in subse-
quent studies in vitro as described below.
Immunoblot Detection of Kifc2 in Mouse Liver
We showed previously that early endocytic vesicles prepared
and the plus-end kinesin Kif5B (Bananis et al., 2003). The pres-
ence of Kifc2 in vesicles prepared from mouse liver was as-
sayed by immunoblot. As seen in Figure 2, Kifc2 was detect-
able in rat liver vesicles and wild-type mouse brain lysate but
not in wild-type mouse liver vesicles. As expected, it was not
detectable in liver or brain from Kifc2 knockout mice. Immu-
noreactivity was removed by preabsorption of antiserum with
the peptide to which it was generated (Figure 2, bottom). These
results indicate that, as distinct from the rat, mouse liver early
endocytic vesicles are not associated with Kifc2.
Preparation of Early Endocytic Vesicles from Mouse Liver
To identify microtubule-based motors that are required for
motility, fluorescent early endocytic vesicles were prepared
Kifc2 knockout mouse hepatocytes. (A) Binding of125I-ASOR to the
surface of hepatocytes was assayed as described in Materials and
Methods. The percentage of initially bound125I-ASOR remaining on
the surface of replicate plates at varied times after incubation at
37°C in hepatocytes from wild-type (closed circles) and Kifc2 knock-
out (open circles) mice is shown. (B) Release of degradation prod-
ucts of125I-ASOR into the medium was assayed as described in
Materials and Methods and is shown as percentage of initially bound
125I-ASOR. Each study was done in triplicate, and the error bar
125I-ASOR binding and degradation in wild-type and
Mouse Early Endosome Motility
Vol. 18, May 2007 1841
from mouse liver 5 min after portal venous injection of 50 ?g
of Texas Red ASOR. The procedure was identical to that
used for rat liver (Bananis et al., 2000, 2003; Murray et al.,
2000; Murray and Wolkoff, 2005). Vesicles were attached to
a glass motility chamber, and ASGPR was visualized by
immunofluorescence. Its distribution on vesicles was com-
pared with that of Texas Red ASOR. As illustrated in Figure
3, 95% of ASOR-containing vesicles isolated from both wild-
type and Kifc2 knockout mice were associated with the
ASGPR, indicating that these vesicles represent a population
of early (presegregation) endocytic vesicles similar to the
early endocytic vesicles that we described previously from
rat liver (Bananis et al., 2000).
Microtubule-based Motility and Fission of Mouse Liver
Early Endocytic Vesicles
These early endocytic vesicles from mouse liver were subjected
to time-lapse fluorescent microscopy to quantify their interac-
tion with and motility along microtubules. In contrast to pre-
vious studies using either rat endocytic vesicles (Bananis et al.,
2000) or vesicles containing herpes simplex virus (Lee et al.,
2006), when mouse early endocytic vesicles were assayed at
have been fixed to the slide began gliding, making the analysis
very difficult (data not shown). We found that this problem of
microtubule movement and detachment was markedly re-
duced when motility assays were conducted on mouse vesicles
at 28°C rather than at 37°C, and subsequent studies were
performed at this lower temperature. On addition of 50 ?M
ATP, 55% of the microtubule-bound wild-type mouse fluores-
cent early endocytic vesicles were motile (Figure 4A and Sup-
plemental Movie 1) with an average nonzero velocity of 0.60 ?
0.04 ?m/s (n ? 52, mean ? SEM). Among these motile vesicles
16% underwent fission (Figure 4B and Supplemental Movie 1).
When the vesicles were preincubated with 5 ?M vanadate, an
inhibitor of the minus-end–directed motor dynein (Kobayashi
et al., 1978; Bananis et al., 2000, 2004; Murray et al., 2000),
motility and fission were essentially unchanged (Figure 4, A
and B). However, pretreatment with 1 mM AMP-PNP, a kine-
sin inhibitor (Vale et al., 1992; Bananis et al., 2000, 2004; Murray
et al., 2000), before ATP addition inhibited vesicle motility and
fission completely (Figure 4, A and B). These results suggest
that microtubule-based motility of mouse early endocytic ves-
icles is mediated by kinesins and not dynein, similar to results
obtained with rat early endocytic vesicles (Bananis et al., 2000).
Early endocytic vesicles prepared from Kifc2 knockout mouse
liver behaved identically (data not shown).
Assessment of Directional Motility
The preceding studies examined total vesicle motility, but
they did not provide information regarding the direction
that vesicles moved on microtubules. To answer this ques-
tion, vesicle motility was assayed on directionally marked
microtubules. These studies showed that 50% of the motile
wild-type early endocytic vesicles moved toward the minus-
end, whereas 42% moved toward the plus-end of microtu-
bules (Figure 4C). The remainder of the vesicles (8%) moved
bidirectionally, indicating activity of both plus- and minus-
end motors on these vesicles. Early endocytic vesicles pre-
pared from Kifc2 knockout mouse liver behaved identically
(Supplemental Movie 2). These data show that early endo-
cytic vesicles prepared from wild-type or Kifc2 knockout
mouse liver move bidirectionally on microtubules and that
this movement is driven by plus- and minus-end–directed
kinesins. Because the vesicles from the knockout mice are
formed after 7.5% SDS-PAGE of liver vesicles or brain lysate from
wild-type or Kifc2 knockout mice as indicated (15 ?g protein/lane).
Rat liver early endocytic vesicles and a lysate prepared from 293T
cells overexpressing FLAG-Kifc2 were used as positive controls.
Antiserum was used at 1:1000 dilution before (top) or after (bottom)
absorption for 3 h at room temperature with 10 ?g/ml peptide
against which it was made.
Immunoblot analysis of Kifc2. Immunoblot was per-
wild-type (top) and Kifc2 knockout (bottom) mouse liver early endocytic vesicles were bound to rhodamine-labeled, Taxol-stabilized
microtubules and visualized in the rhodamine channel (left). Microtubule bound vesicles were incubated with antibody against the ASGPR
followed by Cy2-labeled secondary antibody and visualized in the fluorescein isothiocyanate (FITC) channel (middle). Yellow vesicles in the
merged figure (right) indicate colocalization of ligand (ASOR) and receptor (ASGPR). Bar, 10 ?m.
Colocalization of ASOR and its receptor in mouse liver early endocytic vesicles in vitro. Texas Red-labeled ASOR containing
S. Nath et al.
Molecular Biology of the Cell 1842
devoid of Kifc2, the kinesin that mediates minus-end motil-
ity of rat early endocytic vesicles, we investigated whether
another minus-end kinesin was associated with these vesi-
cles. Aside from Kifc2, only four other minus-end kinesins,
Kifc1, -3, -4, and -5, have been described in mammals (Yang
et al., 1997, 2001b, 2006; Yang and Goldstein, 1998; Navolanic
and Sperry, 2000; Miki et al., 2001, 2003; Noda et al., 2001; Xu
et al., 2002; Yang and Sperry, 2003; Zhang and Sperry, 2004).
Kifc1 and Kifc3 were found to be associated with mem-
brane-bound intracellular organelles (Yang and Goldstein,
1998; Hoang et al., 1999; Noda et al., 2001; Xu et al., 2002;
Yang and Sperry, 2003; Zhang and Sperry, 2004; Yang et al.,
2006), whereas Kifc4 and Kifc5 are primarily active in mito-
sis (Yang et al., 1997; Zhang and Sperry, 2004). As seen in
Figure 5, immunoblot analysis showed that Kifc1 and Kifc3
were both present in liver and brain of wild-type and Kifc2
knockout mice. Further studies were then performed to
determine whether either of these motors was functionally
associated with early endocytic vesicles.
Immunofluorescence Colocalization of Motor Molecules
with Early Endocytic Vesicles
Using specific antibodies, we quantified the association of
the candidate minus-end–directed kinesins Kifc1 and Kifc3
with Texas Red ASOR-loaded early endocytic vesicles pre-
pared from wild-type and Kifc2 knockout mice. Kifc2 anti-
body was used as a control, because it did not detect the
protein on Western blot of mouse vesicle preparations. Cor-
responding studies were also performed using antibodies to
dynein and the plus-end–directed kinesin Kif5B. As seen in
showed that only antibodies to Kifc1 and Kif5B colocalized
with these vesicles. The relatively low degree of colocaliza-
tion of antibodies to dynein, Kifc2, and Kifc3 was similar to
that seen with nonimmune IgG (Figure 6A). Representative
immunofluorescence images showing colocalization of Kifc1
with wild-type and Kifc2 knockout mouse early endocytic
vesicles are seen in Figure 6B. To confirm that Kifc1 is
associated with endocytic vesicles in vivo, overnight cul-
tured mouse hepatocytes were incubated for 5 min at 37°C
with 10 ?g/ml Texas Red ASOR to label early endocytic
vesicles. Cells were then permeabilized by freeze-thaw,
fixed, and incubated with antibody to Kifc1 and Alexa 488-
labeled secondary antibody. As seen in Figure 6C, most of
the ASOR-containing vesicles in these cells were associated
Effects of Motor Antibodies on Mouse Liver Early
Endocytic Vesicle Motility and Fission
To assess which vesicle-associated motors mediated micro-
tubule-based motility, we quantified motility and fission in
and directional motility of early endocytic vesicles from wild-type
mouse liver. Fluorescent early endocytic vesicles were bound to
microtubules. (A) Motility was initiated by addition of 50 ?M ATP
in the absence or presence of 5 ?M vanadate or 1 mM AMP-PNP.
The total number of vesicles counted is indicated above the bars.
*p ? 0.0005 compared with control, and values represent data
compiled from 10 (buffer) and four (vanadate and AMP-PNP) in-
dependent experiments. (B) The fraction of motile vesicles under-
going fission is shown. The total number of motile vesicles exam-
ined is indicated above the bars. (C) Polarity marked microtubules
were prepared and bound to the glass surface of the optical chamber
using DEAE dextran as described in Materials and Methods. Early
endocytic vesicles were then perfused into the chamber. The bars
indicate the percentage of motile vesicles moving toward the plus-
or minus-ends of the microtubules after addition of 50 ?M ATP.
Motility of 36 vesicles from five independent experiments was
examined in this representative study.
Effect of vanadate and AMP-PNP on motility, fission,
were performed after 10% SDS-PAGE of wild-type mouse and Kifc2
knockout mouse brain lysates, rat liver early endocytic vesicles, and
wild-type or Kifc2 knockout mouse liver early endocytic vesicles (25
?g protein/lane) by using Kifc1 (top) or Kifc3 (bottom) antibodies.
Immunoblot analyses of Kifc1 and Kifc3. Immunoblots
Mouse Early Endosome Motility
Vol. 18, May 20071843
from wild-type or Kifc2 knockout mouse livers were perfused into optical chambers and incubated with nonimmune IgG, or dynein, Kifc1,
Kifc2, Kifc3, or Kif5B antibodies followed by incubation with Cy2-labeled secondary antibody. Vesicles were visualized in the rhodamine
channel, and antibody staining was visualized in the FITC channel. The percentage of ASOR-containing vesicles colocalizing with each
antibody is shown. The total number of vesicles examined is indicated above the bars. *p ? 0.0005 compared with nonimmune IgG. (B)
Representative fluorescence micrographs in which colocalization of ASOR-containing vesicles (red) with Kifc1 (green) was examined. The
yellow vesicles in the merged images (right column) indicate colocalization. (C) Overnight cultured mouse hepatocytes were incubated with
10 ?g/ml Texas Red ASOR for 5 min at 37°C. They were then snap-frozen at ?80°C and processed for immunodetection of Kifc1 as described
in Materials and Methods. A bright-field image showing a portion of a hepatocyte (N, nucleus) is in the panel on the left, and vesicles in the
same field containing ASOR or Kifc1 are in the next two panels as indicated. A merged fluorescence image is on the right, and arrows indicate
ASOR-containing vesicles that also contain Kifc1. Bar, 10 ?m.
Immunofluorescence colocalization of motors with endocytic vesicles. (A) Texas Red ASOR-containing early endocytic vesicles
S. Nath et al.
Molecular Biology of the Cell 1844
the presence or absence of specific motor antibodies. Using
this strategy in previous studies, we showed that antibodies
to plus-end–directed conventional kinesin Kif5B and minus-
end–directed kinesin Kifc2 inhibited motility and fission of
early endocytic vesicles prepared from rat liver (Bananis et
al., 2003, 2004). In the present study, preincubation of wild-
type mouse liver early endocytic vesicles with antibody to
Kif5B reduced vesicle motility by ?50% (Figure 7A). Al-
though antibody against Kifc1 did not alter the number of
motile vesicles from either mouse or rat (Figure 7A), the
number of fission events was significantly reduced by ?50%
in the mouse (Figure 7B). There was no effect of this anti-
body on fission of rat vesicles. Antibodies to Kifc2 or Kifc3
had no effect on motility or fission of mouse early endocytic
vesicles (Figure 7). Experiments were also performed to
determine whether Kifc1, in addition to Kifc2, is associated
with rat early endocytic vesicles. Although Kifc1 was
present in the rat liver vesicle preparation (Figure 5), we
found little (18%) colocalization of rat early endocytic vesi-
cles with this motor by immunofluorescence assay. These
results are in contrast to the ?60% colocalization of these
vesicles with Kifc2 that we reported previously (Bananis et
al., 2003, 2004). Consistent with these findings, Kifc1 anti-
body did not have any effect on motility or fission of rat
early endocytic vesicles (Figure 7).
To examine the mechanism by which antibody to Kifc1
inhibited fission but not the number of motile mouse liver
early endocytic vesicles, we determined its effect on direc-
tional motility. As seen in Figure 8, when early endocytic
vesicles from wild-type mouse liver were incubated with
Kifc1 antibody, the proportion of vesicles moving toward
the minus-end of directionally labeled microtubules fell
from 50 to 29%. There was a compensatory increase (from 42
to 71%) of plus-end movement, accounting for the fact that
overall motility of the vesicles was unchanged. These results
suggest that Kifc1 and Kif5B may coexist on a population of
early endocytic vesicles where they act antagonistically. To
measure this directly, the colocalization of Kifc1 and Kif5B
on ASOR-containing mouse vesicles was analyzed by quan-
titative immunofluorescence. Two analyses of the image
data obtained from each microscopy chamber were per-
formed: one analysis to measure the amount of overlap of
Kif5B in the set of vesicles containing Kifc1 and ASOR, and
the other analysis to measure the amount of overlap of Kifc1
in the set of vesicles containing Kif5B and ASOR. As seen in
Table 1 and the representative immunofluorescence colocal-
ization study in Figure 9A, ?90% of the ASOR-containing
vesicles that were associated with Kifc1 were also associated
with Kif5B. Conversely, ?68% of the ASOR-containing ves-
icles that were associated with Kifc1 were also associated
with Kif5B (Table 1). These data indicate that inhibition of
the minus end kinesin Kifc1 results in unopposed Kif5B
motility and fission. Fluorescent ASOR-containing endocytic vesi-
cles from wild-type mouse (black) or rat (gray) liver were bound to
microtubules and incubated for 6 min in the absence or presence of
motor antibodies, as described in Materials and Methods, before
addition of 50 ?M ATP. The percentage of vesicles moving along
microtubules (A) and the percentage of motile vesicles undergoing
fission (B) are shown. The total number of vesicles examined in each
condition is shown in parentheses and represents data obtained
from three to 10 independent experiments. *p ? 0.05 compared with
buffer control by chi-square analysis.
Effect of motor antibodies on early endocytic vesicle
liver early endocytic vesicles. Fluorescent ASOR-containing endo-
cytic vesicles from wild-type mouse liver were bound to polarity
marked microtubules in an optical chamber. Vesicles were incu-
bated for 6 min in the absence or presence of Kifc1 antibody. The
bars indicate the percentage of motile vesicles moving toward the
plus- (black) or minus (gray)-ends of microtubules after addition of
50 ?M ATP. Motility of 36 vesicles was examined in buffer and 42 in
the presence of Kifc1 antibody over five independent experiments
for each condition. *p ? 0.05 compared with control by chi-square
Effect of Kifc1 antibody on directional motility of mouse
Mouse Early Endosome Motility
Vol. 18, May 2007 1845
activity with consequent augmentation of plus-end move-
ment of these vesicles.
Interaction of Kifc1 with Kif5B
Finding that Kifc1 and Kif5B are associated with the same
early endocytic vesicles raises the question as to whether
they interact with each other. Recent studies showed inter-
action of Kif5B (kinesin-1) with dynein (Ligon et al., 2004). To
examine whether Kifc1 interacts with Kif5B, 293T cells that
express Kif5B endogenously (Figure 9B) were transfected
with either FLAG-Kifc1 or FLAG vector. Absorption with
anti-FLAG agarose beads revealed an abundance of Kif5B in
cells transfected with FLAG-Kifc1, but only a small amount
under control conditions with FLAG vector alone (Figure
9B). Corresponding kinesin light chain was also found in the
immunoabsorbate when reprobed with anti KLC antibody
(data not shown). Previous studies suggested that Kif5B
interacted through its light chains with the IC of dynein
(Ligon et al., 2004). Mouse liver expresses KLC2 (Rahman et
al., 1998). To test whether Kifc1 binds to Kif5B through its
light chain, 293T cells were cotransfected with expression
plasmids encoding FLAG-Kifc1 and KLC2. Both proteins
were expressed, but there was no detectable KLC2 in the
FLAG immunoabsorbate, suggesting that these proteins do
not interact (data not shown). These results support the
notion that interaction of Kifc1 and Kif5B requires the Kif5B
heavy chain, although we do not as yet know whether these
proteins interact directly or via a scaffold of other potentially
Receptor-mediated endocytosis is a biologically essential
process in which ligands, including hormones, toxins, and
viruses, interact with specific cell surface receptors and traf-
fic through a series of intracellular vesicular structures along
microtubules (Mellman, 1996; Mukherjee et al., 1997; Murray
and Wolkoff, 2003). The mechanisms coordinating and reg-
ulating this complex process have been the subject of inves-
tigation by many laboratories (Bananis et al., 2003, 2004;
Maxfield and McGraw, 2004; Macia et al., 2006). In recent
Table 1. Simultaneous association of Kifc1 and Kif5B with early
No. of ASOR-positive vesicles
No. in group 1 positive for Kifc1
No. in group 2 positive for Kif5B
% of Kifc1 vesicles with Kif5B
% of ASOR vesicles with KifC1 and Kif5B
No. of ASOR-positive vesicles
No. in group 4 positive for Kif5B
No. in group 5 positive for Kifc1
% of Kif5B vesicles with Kifc1
% of ASOR vesicles with KifC1 and Kif5B
Texas Red ASOR-containing early endocytic vesicles from wild-type
mouse liver were bound to an optical chamber and immunostained
for Kifc1 and Kif5B sequentially as described in Materials and Meth-
ods. In the first image analysis (analysis 1), ASOR-containing vesi-
cles were identified (group 1), and those that also contained Kifc1
(group 2) were quantified. Next, the fraction of the group 2 vesicles
that also contained Kif5B was quantified. Image analysis of ASOR-
containing vesicles was also done in the reverse order (analysis 2).
mouse liver were perfused into optical chambers and incubated with Kifc1 antibody followed by incubation with Alexa 488-labeled
secondary antibody. After washing, the vesicles were incubated with antibody to Kif5B followed by Cy5-labeled secondary antibody, as
described in Materials and Methods. Vesicles were visualized in the rhodamine channel, and antibody staining was visualized in the FITC
(Kifc1) and Cy5 (Kif5B) channels. A typical experiment in which ASOR (red), Kifc1 (green), and Kif5B (blue) were examined is shown.
Vesicles in white in the merged image (right) represent early endocytic vesicles that are associated with both motors simultaneously. Bar, 10
?m. (B) To examine whether Kifc1 and Kif5B can exist in a complex, 293T cells that endogenously express Kif5B were transfected with
FLAG-Kifc1 or FLAG vector alone, and a FLAG immunoprecipitate was examined by immunoblot by using antibody to the kinesin heavy
chain (KHC). Lanes in the immunoblot are cell lysate (input), representing 2% of total volume; material flowing through the anti-FLAG-
agarose beads (FT), representing 2% of total volume; material in the buffer wash (wash), representing 2% of total volume; and material
released from the beads after incubation in sample buffer (beads), representing 50% of bound material.
Interaction of Kifc1 and Kif5B in early endocytic vesicles. (A) Texas Red ASOR-containing early endocytic vesicles from wild-type
S. Nath et al.
Molecular Biology of the Cell1846
studies, we used the hepatocyte-specific asialoglycoprotein
receptor system to produce endocytic vesicles from rat liver
that were loaded with fluorescent ligand (ASOR), and a mi-
croscopy assay to quantify motility and processing of these
vesicles on microtubules in vitro was established (Murray and
Wolkoff, 2005). These studies showed that early endocytic ves-
icles prepared from rat liver move bidirectionally on microtu-
bules, by using the plus- and minus-end–directed kinesins
Kif5B and Kifc2, respectively (Bananis et al., 2003). Based on
these results, we predicted that disruption of Kifc2 function
would reduce endocytic processing of ASOR, and availability
of a Kifc2 knockout mouse provided the opportunity to test
this hypothesis. This mouse model was reported as having
normal embryonic viability and no grossly discernible pheno-
type (Yang et al., 2001a). In conformity with these results, we
mice had normal processing of125I-ASOR and that Kifc2 was
not present in wild-type mouse liver (Figure 2).
These results suggested that in contrast to the rat, Kifc2
plays no role in endocytic vesicle processing in mouse liver.
Previous methods for preparation and study of in vitro
motility of rat liver early endocytic vesicles were adapted in
the present study to mouse liver. As in the rat, Texas Red-
containing vesicles prepared from mice were highly associ-
ated (95%) with the asialoglycoprotein receptor (Figure 3),
indicating that they represent a presegregation population
of early endocytic vesicles (Bananis et al., 2000). These vesi-
cles bound to microtubules in vitro and moved bidirection-
ally after ATP addition. Motility of early endocytic vesicles
prepared from Kifc2 knockout mice was indistinguishable
from that of wild-type mice. There was no evidence that the
minus-end motility of these vesicles was due to substitution
of dynein for Kifc2. Specifically, there was no inhibition of
vesicle motility in the presence of 5 ?M vanadate (Figure 4),
a treatment that inhibits dynein-mediated motility (Bananis
et al., 2000; Sarkar et al., 2006), and there was no immuno-
colocalization of dynein with fluorescent ASOR-containing
vesicles (Figure 6A). Rather, as was found in studies of early
endocytic vesicles from the rat, all motility in both wild-type
and Kifc2 knockout mouse vesicles was inhibited by 1 mM
AMP-PNP, an inhibitor of kinesin based motility (Vale et al.,
1992; Bananis et al., 2000; Murray et al., 2000). A recent study
in which HeLa cells were loaded with fluorescent epidermal
growth factor for 2–3 min and followed for as long as 30 min
was interpreted as showing a role for dynein in processing
of early endocytic vesicles (Driskell et al., 2007), in contrast to
other studies (Nielsen et al., 1999; Bananis et al., 2003. 2004).
As processing of endocytic vesicles is dynamic (Bananis et
al., 2004; Sarkar et al., 2006), it is possible that these vesicles
had already passed a transition point toward late vesicles,
which we have shown are associated with dynein (Bananis
et al., 2004). Alternatively, they could represent a different
population of early/recycling endocytic vesicles such as
those that contain the bile acid transporter sodium tauro-
cholate cotransporting polypeptide (ntcp) (Sarkar et al.,
2006). These ntcp-containing vesicles cycle to and from the
basolateral plasma membrane of hepatocytes, by using
Kif5B and dynein (Sarkar et al., 2006).
Because these data indicated the presence of a minus-end–
directed kinesin on mouse early endocytic vesicles and Kifc2
was absent, we considered the presence of other minus-end
kinesins, of which only four others have been described
previously (Yang et al., 1997, 2001a,b, 2006; Navolanic and
Sperry, 2000; Noda et al., 2001; Miki et al., 2003; Yang and
Sperry, 2003; Zhang and Sperry, 2004). Within this group,
Kifc1 and Kifc3 were associated with membranous or-
ganelles (Yang et al., 1997; Navolanic and Sperry, 2000; Noda
et al., 2001; Yang et al., 2001b, 2006; Yang and Sperry, 2003;
Zhang and Sperry, 2004), whereas Kifc4 and Kifc5 were seen
in mitosis where they play a role in chromosome movement
(Yang et al., 1997; Zhang and Sperry, 2004). Consequently,
we looked for the presence of Kifc1 and Kifc3 on mouse
early endocytic vesicles. Immunoblot analysis (Figure 5)
showed the presence of both Kifc1 and Kifc3 in mouse
vesicle preparations from wild-type and Kifc2 knockout
mice. These preparations consist of a mixture of vesicles
containing endocytosed fluorescent ligand and other unla-
beled vesicles. We found that only Kifc1, but not Kifc3, was
substantially associated with the ASOR-containing vesicles
as revealed by immunofluorescence colocalization (Figure
6). Association of Kifc3 with ASOR-containing vesicles was
at background levels, although it was present on unidenti-
fied vesicles that did not contain Texas Red ASOR (data not
shown). Approximately 80% of mouse early endocytic ves-
icles were also associated with the plus-end kinesin Kif5B,
similar to results in rat vesicles (Bananis et al., 2000, 2003,
The findings that ?50% of mouse early endocytic vesicles
are associated with Kifc1 and ?80% with Kif5B suggest that
there is a population of vesicles that must be associated
simultaneously with both motors. As seen in Table 1, ?90%
of vesicles that were associated with Kifc1 were also associ-
ated with Kif5B. Of the total population of vesicles contain-
ing ASOR, approximately half were associated simulta-
neously with both motors. This is in agreement with two
observations. First, that some vesicles moving in one direc-
tion along a microtubule stop and then move in the other
direction. Second, that plus end motility is increased when
minus-end motility is inhibited by incubation of vesicles
with antibody to Kifc1 (Figure 8). These data are consistent
with the possibility that these motors may be part of a yet to
be elucidated protein complex that mediates their coordi-
nate regulation, as has been suggested for vesicle-associated
dynein and plus-end kinesins (Brady et al., 1990; Stenoien
and Brady, 1997; Waterman-Storer et al., 1997; Ligon et al.,
2004). This view is supported in the present study by the
finding that Kifc1 and Kif5B can interact with each other as
shown by immunoabsorption of native Kif5B by FLAG-
Kifc1 expressed in 293T cells (Figure 9B). Whether these
motors interact directly or through a complex of potential
regulatory proteins will need to be clarified in future studies.
It is of interest that in contrast to results with antibody to
Kifc1, overall motility of vesicles is reduced after incubation
with antibody to Kif5B. This is in agreement with results
observed in several previous studies (Brady et al., 1990;
Martin et al., 1999; Ligon et al., 2004; Theiss et al., 2005; Sarkar
et al., 2006), suggesting that Kif5B activity is dominant over
other motor activities (Sarkar et al., 2006). The finding that
fission of vesicles is reduced when either Kifc1 or Kif5B
activity is inhibited by antibody also suggests that the op-
posing forces resulting from activity of both motors are
required for fission to occur.
Although Kifc1 and Kifc2 are both present in rat liver
vesicle preparations (Figure 5), our data indicate that only
Kifc2 is used for motility of ASOR-containing rat early en-
docytic vesicles (Figure 7; Bananis et al., 2003). Thus minus-
end–directed motility of early endocytic vesicles is mediated
by Kifc2 in the rat and Kifc1 in the mouse. These are genet-
ically distinct proteins with little homology (Saito et al.,
1997). Specific regions of a number of kinesins that interact
with and bind to cargo have been identified previously
(Hirokawa, 1998; Verhey et al., 2001; Smith et al., 2006). In
particular, previous studies of Kifc1 identified a 19-amino
acid sequence that is required for binding to membrane-
Mouse Early Endosome Motility
Vol. 18, May 20071847
bounded organelles (Zhang and Sperry, 2004). This Kifc1-
specific sequence has no corresponding region in Kifc2,
which presumably has its own unique, although not yet
characterized, organelle-interacting sequence. The endocytic
vesicle-associated proteins that interact with these binding
regions on kinesins are not known, but differences in the
protein constituents of rat compared with mouse early en-
docytic vesicles are likely to be a primary factor in the
species difference in minus-end–directed kinesin recruit-
This study showed that coordinated activity of plus- and
minus-end–directed kinesins is essential for motility and
processing early endocytic vesicles. Little is known about
the role of minus-end kinesins in vesicle trafficking, al-
though their importance in mitosis is clear (Zhang and
Sperry, 2004; Goshima et al., 2005; Christodoulou et al., 2006).
There are homologues of Kifc1 and Kifc2 in other species,
such as Caenorhabditis elegans (Robin et al., 2005) and Saccha-
romyces cerevisiae (Maddox, 2005), but their roles in vesicle
trafficking have not been examined. The present study
shows that function of these motors may be substantially
different from species to species, likely depending on differ-
ential interaction with other vesicle-associated proteins. It is
also of interest that early endocytic vesicles do not use
dynein for minus-end motility. Rather, our previous studies
showed that dynein mediates minus-end motility of late
endocytic vesicles (Bananis et al., 2004). Presumably, inter-
action with dynein requires binding to specific vesicle-asso-
ciated proteins that are not present on early endocytic ves-
icles. Proteomic analysis of specific populations of endocytic
vesicles may help to identify and characterize these proteins
(Bananis et al., 2004).
We thank Dr. Lawrence S.B. Goldstein for kindly providing Kifc2 knockout
mice, Kifc2 cDNA, and antibody to Kif5B and Dr. Erika L.F. Holzbaur for
providing KLC2 cDNA. We also thank Dr. Brigid Joseph (Albert Einstein
College of Medicine) and David S. Neufeld (Albert Einstein College of Med-
icine) for assistance with hepatocyte isolation. This work was supported by
National Institutes of Health Grants DK-41918 (to A.W.W.) and GM-60628 (to
Bananis, E., Murray, J. W., Stockert, R. J., Satir, P., and Wolkoff, A. W. (2000).
Microtubule and motor-dependent endocytic vesicle sorting in vitro. J. Cell
Biol. 151, 179–186.
Bananis, E., Murray, J. W., Stockert, R. J., Satir, P., and Wolkoff, A. W. (2003).
Regulation of early endocytic vesicle motility and fission in a reconstituted
system. J. Cell Sci. 116, 2749–2761.
Bananis, E., Nath, S., Gordon, K., Satir, P., Stockert, R. J., Murray, J. W., and
Wolkoff, A. W. (2004). Microtubule-dependent movement of late endocytic
vesicles in vitro: requirements for dynein and kinesin. Mol. Biol. Cell 15,
Brady, S. T., Pfister, K. K., and Bloom, G. S. (1990). A monoclonal antibody
against kinesin inhibits both anterograde and retrograde fast axonal transport
in squid axoplasm. Proc. Natl. Acad. Sci. USA 87, 1061–1065.
Christodoulou, A., Lederer, C. W., Surrey, T., Vernos, I., and Santama, N.
(2006). Motor protein KIFC5A interacts with Nubp1 and Nubp2, and is
implicated in the regulation of centrosome duplication. J. Cell Sci. 119, 2035–
Driskell, O. J., Mironov, A., Allan, V. J., and Woodman, P. G. (2007). Dynein
is required for receptor sorting and the morphogenesis of early endosomes.
Nat. Cell Biol. 9, 113–120.
Goltz, J. S., Wolkoff, A. W., Novikoff, P. M., Stockert, R. J., and Satir, P. (1992).
A role for microtubules in sorting endocytic vesicles in rat hepatocytes. Proc.
Natl. Acad. Sci. USA 89, 7026–7030.
Goshima, G., Nedelec, F., and Vale, R. D. (2005). Mechanisms for focusing
mitotic spindle poles by minus end-directed motor proteins. J. Cell Biol. 171,
Hanlon, D. W., Yang, Z., and Goldstein, L. S. (1997). Characterization of
KIFC2, a neuronal kinesin superfamily member in mouse. Neuron 18, 439–
Harford, J., Bridges, K., Ashwell, G., and Klausner, R. D. (1983a). Intracellular
dissociation of receptor-bound asialoglycoproteins in cultured hepatocytes. A
pH-mediated nonlysosomal event. J. Biol. Chem. 258, 3191–3197.
Harford, J., Wolkoff, A. W., Ashwell, G., and Klausner, R. D. (1983b). Monen-
sin inhibits intracellular dissociation of asialoglycoproteins from their recep-
tor. J. Cell Biol. 96, 1824–1828.
Higgins, M. K., and McMahon, H. T. (2002). Snap-shots of clathrin-mediated
endocytosis. Trends Biochem. Sci. 27, 257–263.
Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mech-
anism of organelle transport. Science 279, 519–526.
Hoang, E., Bost-Usinger, L., and Burnside, B. (1999). Characterization of a
novel C-kinesin (KIFC3) abundantly expressed in vertebrate retina and RPE.
Exp. Eye Res. 69, 57–68.
Kobayashi, T., Martensen, T., Nath, J., and Flavin, M. (1978). Inhibition of
dynein ATPase by vanadate, and its possible use as a probe for the role of
dynein in cytoplasmic motility. Biochem. Biophys. Res. Commun. 81, 1313–
Lawrence, C. J. et al. (2004). A standardized kinesin nomenclature. J. Cell Biol.
Lee, G. E., Murray, J. W., Wolkoff, A. W., and Wilson, D. W. (2006). Recon-
stitution of herpes simplex virus microtubule-dependent trafficking in vitro.
J. Virol. 80, 4264–4275.
Ligon, L. A., Tokito, M., Finklestein, J. M., Grossman, F. E., and Holzbaur,
E. L. (2004). A direct interaction between cytoplasmic dynein and kinesin I
may coordinate motor activity. J. Biol. Chem. 279, 19201–19208.
Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen,
T. (2006). Dynasore, a cell-permeable inhibitor of dynamin. Dev. Cell 10,
Maddox, P. S. (2005). Microtubules: Kar3 eats up the track. Curr. Biol. 15,
Marsh, M., and McMahon, H. T. (1999). The structural era of endocytosis.
Science 285, 215–220.
Martin, M., Iyadurai, S. J., Gassman, A., Gindhart, J. G., Jr., Hays, T. S., and
Saxton, W. M. (1999). Cytoplasmic dynein, the dynactin complex, and kinesin
are interdependent and essential for fast axonal transport. Mol. Biol. Cell 10,
Maxfield, F. R., and McGraw, T. E. (2004). Endocytic recycling. Nat. Rev. Mol.
Cell Biol. 5, 121–132.
Mellman, I. (1996). Endocytosis and molecular sorting. Annu. Rev. Cell Dev.
Biol. 12, 575–625.
Miki, H., Okada, Y., and Hirokawa, N. (2005). Analysis of the kinesin super-
family: insights into structure and function. Trends Cell Biol. 15, 467–476.
Miki, H., Setou, M., and Hirokawa, N. (2003). Kinesin superfamily proteins
(KIFs) in the mouse transcriptome. Genome Res. 13, 1455–1465.
Miki, H., Setou, M., Kaneshiro, K., and Hirokawa, N. (2001). All kinesin
superfamily protein, KIF, genes in mouse and human. Proc. Natl. Acad. Sci.
USA 98, 7004–7011.
Mukherjee, S., Ghosh, R. N., and Maxfield, F. R. (1997). Endocytosis. Physiol.
Rev. 77, 759–803.
Murray, J. W., Bananis, E., and Wolkoff, A. W. (2000). Reconstitution of
ATP-dependent movement of endocytic vesicles along microtubules in vitro:
an oscillatory bidirectional process. Mol. Biol. Cell 11, 419–433.
Murray, J. W., Bananis, E., and Wolkoff, A. W. (2002). Immunofluorescence
microchamber technique for characterizing isolated organelles. Anal. Bio-
chem. 305, 55–67.
Murray, J. W., and Wolkoff, A. W. (2003). Roles of the cytoskeleton and motor
proteins in endocytic sorting. Adv. Drug Deliv. Rev. 55, 1385–1403.
Murray, J. W., and Wolkoff, A. W. (2005). Assay of Rab4-dependent trafficking
on microtubules. Methods Enzymol. 403, 92–107.
Navolanic, P. M., and Sperry, A. O. (2000). Identification of isoforms of a
mitotic motor in mammalian spermatogenesis. Biol. Reprod. 62, 1360–1369.
Nielsen, E., Severin, F., Backer, J. M., Hyman, A. A., and Zerial, M. (1999).
Rab5 regulates motility of early endosomes on microtubules. Nat. Cell Biol. 1,
S. Nath et al.
Molecular Biology of the Cell 1848
Noda, Y., Okada, Y., Saito, N., Setou, M., Xu, Y., Zhang, Z., and Hirokawa, N.
(2001). KIFC3, a microtubule minus end-directed motor for the apical trans-
port of annexin XIIIb-associated Triton-insoluble membranes. J. Cell Biol. 155,
Novikoff, P. M., Cammer, M., Tao, L., Oda, H., Stockert, R. J., Wolkoff, A. W.,
and Satir, P. (1996). Three-dimensional organization of rat hepatocyte cy-
toskeleton: relation to the asialoglycoprotein endocytosis pathway. J. Cell Sci.
Perrais, D., and Merrifield, C. J. (2005). Dynamics of endocytic vesicle cre-
ation. Dev. Cell 9, 581–592.
Rahman, A., Friedman, D. S., and Goldstein, L. S. (1998). Two kinesin light
chain genes in mice. Identification and characterization of the encoded pro-
teins. J. Biol. Chem. 273, 15395–15403.
Robin, G., DeBonis, S., Dornier, A., Cappello, G., Ebel, C., Wade, R. H.,
Thierry-Mieg, D., and Kozielski, F. (2005). Essential kinesins: characterization
of Caenorhabditis elegans KLP-15. Biochemistry 44, 6526–6536.
Saito, N., Okada, Y., Noda, Y., Kinoshita, Y., Kondo, S., and Hirokawa, N.
(1997). KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily
motor for dendritic transport of multivesicular body-like organelles. Neuron
Samuelson, A. C., Stockert, R. J., Novikoff, A. B., Novikoff, P. M., Saez, J. C.,
Spray, D. C., and Wolkoff, A. W. (1988). Influence of cytosolic pH on receptor-
mediated endocytosis of asialoorosomucoid. Am. J. Physiol. 254, C829–C838.
Sarkar, S., Bananis, E., Nath, S., Anwer, M. S., Wolkoff, A. W., and Murray,
J. W. (2006). PKCzeta is required for microtubule-based motility of vesicles
containing the ntcp transporter. Traffic 7, 1078–1091.
Smith, M. J., Pozo, K., Brickley, K., and Stephenson, F. A. (2006). Mapping the
GRIF-1 binding domain of the kinesin, KIF5C, substantiates a role for GRIF-1
as an adaptor protein in the anterograde trafficking of cargoes. J. Biol. Chem.
Stenoien, D. L., and Brady, S. T. (1997). Immunochemical analysis of kinesin
light chain function. Mol. Biol. Cell 8, 675–689.
Stockert, R. J., Haimes, H. B., Morell, A. G., Novikoff, P. M., Novikoff, A. B.,
Quintana, N., and Sternlieb, I. (1980). Endocytosis of asialoglycoprotein-
enzyme conjugates by hepatocytes. Lab. Investig. 43, 556–563.
Theiss, C., Napirei, M., and Meller, K. (2005). Impairment of anterograde and
retrograde neurofilament transport after anti-kinesin and anti-dynein anti-
body microinjection in chicken dorsal root ganglia. Eur. J. Cell Biol. 84, 29–43.
Treichel, U., Paietta, E., Poralla, T., Meyer zum Buschenfelde, K. H., and
Stockert, R. J. (1994). Effects of cytokines on synthesis and function of the
hepatic asialoglycoprotein receptor. J. Cell Physiol. 158, 527–534.
Vale, R. D., Malik, F., and Brown, D. (1992). Directional instability of micro-
tubule transport in the presence of kinesin and dynein, two opposite polarity
motor proteins. J. Cell Biol. 119, 1589–1596.
Verhey, K. J., Meyer, D., Deehan, R., Blenis, J., Schnapp, B. J., Rapoport, T. A.,
and Margolis, B. (2001). Cargo of kinesin identified as jip scaffolding proteins
and associated signaling molecules. J. Cell Biol. 152, 959–970.
Waterman-Storer, C. M., Karki, S. B., Kuznetsov, S. A., Tabb, J. S., Weiss, D. G.,
Langford, G. M., and Holzbaur, E. L. (1997). The interaction between cyto-
plasmic dynein and dynactin is required for fast axonal transport. Proc. Natl.
Acad. Sci. USA 94, 12180–12185.
Wolkoff, A. W., Klausner, R. D., Ashwell, G., and Harford, J. (1984). Intracel-
lular segregation of asialoglycoproteins and their receptor: a prelysosomal
event subsequent to dissociation of the ligand-receptor complex. J. Cell Biol.
Xu, Y., Jones, B. E., Neufeld, D. S., and Czaja, M. J. (1998). Glutathione
modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor
toxicity. Gastroenterology 115, 1229–1237.
Xu, Y., Takeda, S., Nakata, T., Noda, Y., Tanaka, Y., and Hirokawa, N. (2002).
Role of KIFC3 motor protein in Golgi positioning and integration. J. Cell Biol.
Yang, W. X., Jefferson, H., and Sperry, A. O. (2006). The molecular motor
KIFC1 associates with a complex containing nucleoporin NUP62 that is
regulated during development and by the small GTPase RAN. Biol. Reprod.
Yang, W. X., and Sperry, A. O. (2003). C-Terminal kinesin motor KIFC1
participates in acrosome biogenesis and vesicle transport. Biol. Reprod. 69,
Yang, Z., and Goldstein, L. S. (1998). Characterization of the KIF3C neural
kinesin-like motor from mouse. Mol. Biol. Cell 9, 249–261.
Yang, Z., Hanlon, D. W., Marszalek, J. R., and Goldstein, L. S. (1997). Identi-
fication, partial characterization, and genetic mapping of kinesin-like protein
genes in mouse. Genomics 45, 123–131.
Yang, Z., Roberts, E. A., and Goldstein, L. S. (2001a). Functional analysis of
mouse C-terminal kinesin motor KifC2. Mol. Cell Biol. 21, 2463–2466.
Yang, Z., Xia, C., Roberts, E. A., Bush, K., Nigam, S. K., and Goldstein, L. S.
(2001b). Molecular cloning and functional analysis of mouse C-terminal ki-
nesin motor KifC3. Mol. Cell Biol. 21, 765–770.
Zhang, Y., and Sperry, A. O. (2004). Comparative analysis of two C-terminal
kinesin motor proteins: KIFC1 and KIFC5A. Cell Motil. Cytoskeleton 58,
Mouse Early Endosome Motility
Vol. 18, May 2007 1849