# 2008 The Authors

Journal compilation # 2008 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2008.00725.xTraffic 2008; 9: 833–847Blackwell Munksgaard

Single Vesicle Analysis of Endocytic Fission onMicrotubules In Vitro

John W. Murray1,2,†,*, Souvik Sarkar1,2,† and

Allan W. Wolkoff1,2

1Marion Bessin Liver Research Center and Departmentof Medicine, and Division of Hepatology, Albert EinsteinCollege of Medicine, 1300 Morris Park Avenue,Bronx, NY 10461, USA2Department of Anatomy and Structural Biology,Albert Einstein College of Medicine, 1300 MorrisPark Avenue, Bronx, NY 10461, USA*Corresponding author: John W. Murray,jwmurray@aecom.yu.edu†These authors contributed equally to this work.

Following endocytosis, internalized molecules are found

within intracellular vesicles and tubules that move along

the cytoskeleton and undergo fission, as demonstrated

here using primary cultured rat hepatocytes. Although

the use of depolymerizing drugs has shown that the

cytoskeleton is not required to segregate endocytic

protein, many studies suggest that the cytoskeleton

is involved in the segregation of protein in normal cells.

To investigate whether cytoskeletal-based movement

results in the segregation of protein, we tracked the

contents of vesicles during in vitro microscopy assays.

These studies showed that the addition of ATP causes

fission of endocytic contents along microtubules, result-

ing in the segregation of proteins that are targeted for

different cellular compartments. The plasma membrane

proteins, sodium (Naþ) taurocholate cotransporting

polypeptide (ntcp) and transferrin receptor, segregated

from asialoorosomucoid (ASOR), an endocytic ligand that

is targeted for degradation. Epidermal growth factor

receptor, which is degraded, and the asialoglycoprotein

receptor, which remains partially bound to ASOR, segre-

gated less efficiently from ASOR. Vesicles containing

ntcp and transferrin receptor had reduced fission in the

absence of ASOR, suggesting that fission is regulated to

allow proteins to segregate. A single round of fission

resulted in 6.5-fold purification of ntcp from ASOR, and

25% of the resulting vesicles were completely depleted of

the endocytic ligand.

Key words: asialoglycoprotein receptor, endosome,

fission, microtubules, sorting, transferrin receptor

Received 18 July 2007, revised and accepted for publica-

tion 11 February 2008, uncorrected manuscript published

online 16 February 2008, published online 18 March 2008

Proteins at the plasma membrane are continuously endo-

cytosed and transported to intracellular early (sorting)

endosomes. From early endosomes, proteins are typically

sorted either to late endosomes, for subsequent degrada-

tion, or to recycling endosomes, from which they traffic

back to the plasma membrane (1,2). Coat proteins and

adaptor proteins appear to function in protein sorting and

budding at the endosome (3,4). However, examination

of endosomal protein within mammalian cells by light

microscopy reveals dramatic cytoskeletal-based trafficking,

tubule formation and fission (5–7), while examination by

electron microscopy reveals tubular extensions corres-

ponding to sites of protein sorting (8,9). Microtubule motor

proteins are involved in the movement of endocytic

proteins from the early/sorting endosomes to the perinu-

clear recycling vesicles and late endosomes (10,11), and it

has been shown that microtubule depolymerization can

delay protein degradation and block apical recycling and

transcytosis (12–15). However, it has also been shown in

cultured cells that the depolymerization of microtubules

does not block protein recycling (16,17). Therefore, the

precise role of microtubules in protein segregation is

unclear. The studies presented here address this issue

by examining how microtubule-based movement of endo-

cytic material that is seen by time-lapse microscopy of

living cells contributes to protein sorting and how this

sorting manifests itself at the level of an individual fission

event. Few reports have quantified whether cytoskeletal-

based fission can result in specific segregation of different

proteins. In contrast, the mechanisms of endocytic vesicle

fusion and the importance of cellular SNARE protein

machinery have been well established, in part because of

reconstitution of fusion in vitro (18,19). It has been shown

that fission can be quantitatively analyzed in vitro using

time-lapse microscopy (20). This report broadens those

studies with an in-depth analysis of fission and the

behavior of multiple proteins in order to determine

whether studies of single vesicles can provide an import-

ant tool for understanding the underlying mechanisms of

protein sorting.

Four integral membrane proteins were used in these

studies: transferrin receptor, asialoglycoprotein receptor

(ASGPR), epidermal growth factor receptor (EGFR) and

sodium (Naþ) taurocholate cotransporting polypeptide

(ntcp) as well as the lumenal markers, asialoorosomucoid

(ASOR) and fluorescent dextran. The transferrin receptor

and ASGPR are well-studied integral membrane proteins

that undergo recycling back to the cell surface once

internalized (21,22). In contrast, a significant portion of

EGFR is transported to multivesicular bodies and degraded

once it is internalized (23,24). Ntcp is a hepatocyte-specific

bile acid transporter that localizes to the plasmamembrane

as well as a subplasma membrane intracellular pool

(25,26). ASOR is a ligand for ASGPR and once internalized

www.traffic.dk 833

undergoes dissociation followed by sorting to the lysosomes

where it is degraded (12). Dextran is a soluble pinocytic

marker that enters cells in the fluid phase (27). These

markers were selected in order to study the behavior of

molecules that are targeted for either degradation or

recycling during individual fission events that occur along

microtubules.

Results

Vesicle motility and fission in primary hepatocytes

Previous studies have documented that early endosomes,

late endosomes and lysosomes undergo microtubule-

based movement during the processing of endocytic

contents (7,28,29). However, it has been difficult to

demonstrate how this movement contributes to metabolic

functions such as the segregation of protein. To confirm

that hepatocytes display microtubule-based movement of

their early endosomes, hepatocytes were incubated with

fluorescent endocytic ligand, ASOR, in the presence or

absence of the microtubule-depolymerizing drug, nocoda-

zole. Time-lapse fluorescent microscopy revealed that

ASOR is present within intracellular vesicles after 5 min

of uptake and that these undergo a complicated display of

fusion, fission and back-and-forth movement (Movie S1).

This is seen as bright streaks in the time-lapse overlay in

the top panel of Figure 1A. Treatment of cells with

nocodazole suspended nearly all long-distance motility

events (Movie S2), as seen by the lack of bright streaks

in the bottom panel of Figure 1A. Some smaller move-

ments and vibrating activities were still observed, indicat-

ing that endosomes were not rigidly fixed and that cells

were still capable of movement that did not depend on

microtubules.

We next asked whether vesicles containing proteins that

are targeted to different cellular locations could be seen

undergoing fission while moving on microtubules. We also

examined whether these fission events resulted in the

segregation of one protein from the other. Hepatocytes

were incubated with Alexa-488-labeled transferrin and

Texas red-labeled ASOR and subsequently imaged in

two fluorescence channels (Figure 1B). Transferrin re-

mains bound to its receptor during cycles of endocytosis

and recycling where it moves from the cell surface into

endosomes and then back to the cell surface (21). How-

ever, ASOR is taken up by the ASGPR and released within

endocytic compartments where it subsequently traffics to

the lysosomes and is degraded (30). As seen in Movie S3

and zoomed in Movies S5 and S6 (Movie S4 contains the

corresponding bright-field images), vesicles containing

both markers were highly motile and could be seen to

undergo fission. Still images fromMovie S5 demonstrating

fission are shown in Figure 1C.

These data indicate that a single vesicle can contain two

different proteins and undergo microtubule-based move-

ment and fission. However, these findings do not imply

that these events can lead to the overall segregation of

these proteins from each other. Whereas many studies

indicate that microtubule-based trafficking contributes to

endocytic protein sorting and the recycling of protein

(10,31), other studies have demonstrated that depolymer-

ization of the microtubule cytoskeleton does not block

protein recycling (16,17). In an attempt to address these

issues, we initiated studies to quantify the overall redistri-

bution of proteins during single vesicle fission events within

living cells. However, varying background fluorescence,

photobleaching and the complex, three-dimensional motion

of the vesicles prohibited accurate quantification of protein

using live cell imaging techniques. Given these difficulties,

we instead took advantage of an in vitro assay that has

been developed to analyze the microtubule motor proteins

that are involved in endocytic vesicle traffic (32,33).

Reconstitution of vesicle fission in vitro

A standardized in vitromotility assay was used to generate

the data below. Similar assays have been used to identify

and purify motor proteins, to characterize mutations of

motor proteins and to show that organelles use specific

sets of motor proteins and regulatory factors for cellular

transport (33–36). In brief, endocytic vesicles are isolated

by homogenization of rat liver followed by sucrose step-

gradient centrifugation. Vesicles are then stored as single-

use, frozen aliquots. Small volume (3 mL) glass microscope

chambers are constructed from glass slides, and micro-

tubules are polymerized from fluorescent tubulin and

attached to the bottom of the chambers. Endocytic

vesicles are thawed and added to the chambers where

they attach to the microtubules, and the unattached

contents are washed away. Fifty micromolar ATP is added

to the chambers to initiate vesicle motility, and this is

captured by high-resolution, wide-field, multi-fluorescence

channel, time-lapse microscopy. Prior to ATP addition,

chambers containing endocytic vesicles that are attached

to microtubules are stored in buffer on ice where they

remain active in motility assays for more than 3 h.

Two approaches are used to identify specific proteins

within the endocytic vesicles: (i) the hepatic portal vein is

injected with fluorescent ligand, and hepatocytes are

allowed to take up the ligand prior to homogenization of

the liver and (ii) the endosomes are labeled by immuno-

fluorescence within the microscope chambers after they

have bound to microtubules. Unless specifically noted, all

the biochemical preparations of vesicles in the following

experiments contain fluorescent-labeled ASOR that has

been injected into the hepatic portal vein 5 min prior to liver

homogenization. The red fluorescent vesicles within the

preparation represent ‘early’ endocytic ASOR as they

contain both ligand and receptor (20). This preparation also

contains a multitude of other vesicles that copurify with

these ASOR-containing vesicles. As described previously,

these vesicles bind to microtubules and move in the

834 Traffic 2008; 9: 833–847

Murray et al.

presence of ATPwithout the addition of exogenous protein

or cytosol (20,32,37). In addition, the moving vesicles

undergo fission. As in living cells, fission is observed as

the separation of a single region, or spot, of fluorescence.

Unlike in living cells, vesicles in vitro remain within the

focal plane during movement, and the history of all move-

ments can be tracked. This facilitates the differentiation of

fission from other motile events, such as the movement of

two vesicles in opposite directions (e.g. vesicle crossing).

Also unlike in living cells, vesicle motility by these techniques

occurs in a single burst that lasts approximately 90 sec-

onds. Subsequently, vesicles detach from microtubules

Figure 1: Legend on next page.

Traffic 2008; 9: 833–847 835

Endocytic Segregation In Vitro

or stop moving, presumably as a result of ATP hydrolysis

and depletion (32) or possibly because of changes in

protein phosphorylation or other factors (26). This relatively

short burst of movement simplifies data collection and

analysis.

Figure 1C (right panels) presents an example of fission

occurring in vitro. A vesicle containing ASOR (red) and

stained with antibody to the transferrin receptor (green) is

attached to a microtubule (white arrow). ATP was added

to the chamber, and after 21 seconds, a daughter vesicle

containing only transferrin receptor emerges, moving

to the right. A portion of the transferrin receptor remains

with the mother vesicle. Note that the size of the vesicle

and duration of fission are similar for the examples in the

living cell and in vitro (Figure 1C), that is, the apparent

diameter for both vesicles is approximately 1 mm and the

separation into two vesicles takes less than 10 seconds of

real time. Movies S7 and S8 provide additional examples of

fission events between ASOR and ntcp, and ASOR, ntcp

and transferrin receptor, respectively.

Fission is not the result of the movement of

overlapped vesicles

It is possible that fission observed during in vitro microtu-

bule motility assays results from the separation of over-

lapping or aggregated vesicles. Previous studies have

shown that fission is not reduced by diluting the vesicles

prior to attachment to microtubules (20), indicating that

fission is not the result of overlapped, unconnected

vesicles. To determine whether the vesicle isolation pro-

cedure produces aggregated vesicles, livers from rats that

had endocytosed either Texas red ASOR (red) or Alexa-

labeled ASOR (green) were homogenized together and

vesicles were isolated using the standard protocol. Any

vesicle that aggregates during this process would be

revealed by overlap of red and green fluorescence. The

degree of overlap was assessed in the presence and

absence of microtubules, and the level of ATP-induced

motility and fission was also measured. Images from these

experimentsareshown inFigure 1D.Of the961microtubule-

bound vesicles scored, 671 were green and 290 were red,

and of these, 32 (3.3%) showed some overlap of red and

green fluorescence. Of the overlapped vesicles, only three

underwent separation of green and red during motility

experiments. Thus, the clustering of red and green

vesicles was rare, and those that did cluster did not show

a high level of separation upon addition of ATP.

Vesicle motility and fission varies as a function of

protein constituents

In order to measure the segregation of protein during

microtubule-based fission, we first sought to characterize

the amount of motility and fission of vesicles that con-

tained different endocytic proteins. Several groups have

demonstrated that different populations of endocytic

vesicles have different amounts of motility (6,38). The

endocytic vesicle preparation used in these studies con-

tains the endocytic marker, ASOR, as well as numerous

other vesicles (11,32). Because motor proteins, coat

proteins or other regulatory proteins have the potential to

exchange between vesicles during their purification, it was

possible that all vesicles would display an identical level of

motility and fission in vitro. Alternatively, it was possible

that the amount of fission for some vesicles would be too

low to allow quantification of protein segregation.

We performed in vitro vesicle motility assays and sub-

sequently measured the level of motility and fission for

vesicles that contained the endocytic ligand, ASOR. Motil-

ity is defined as the per cent of microtubule-bound vesicles

that undergo movement, while fission is defined as the per

cent of motile vesicles that undergo fission. Twenty-one

per cent of microtubule-bound vesicles that contained

ASOR were motile (n ¼ 67), and 14% of these underwent

fission (Figure 2). These results are very similar to those

Figure 1: Movement and fission of endocytic vesicles in hepatocytes and in vitro. A) Control or nocodazole-treated hepatocytes

were allowed to endocytose fluorescent ASOR for 5 min. Cells werewashed and imaged by time-lapsemicroscopy for 1- to 1.5-min, 8 min

following the wash. The left panels show the first frame of the movie, and the right panels shows an overlay (maximum intensity

projection) of all movie frames. Arrows point to fluorescence streaks that indicate the movement of the internalized ASOR. These streaks

were absent in nocodazole-treated cells, but short-distance movement was observed as broadening of the fluorescence spots. B) Control

hepatocytes were allowed to endocytose Texas red ASOR (red) and Alexa-488 transferrin (Tfn) (green) and washed, imaged and displayed

as described above. Both ligands showed prominent long-distance movement as indicated by the streaks (arrows). The yellow box

indicates the region of the cell that is magnified for the subsequent figure. C) On the left, fission is observed in a living hepatocyte (from

Movie S5, an additional example is seen in Movie S6), while on the right, fission is observed in vitrowithin a microscope chamber. Arrows

mark the location of ‘mother’ vesicles, while arrowheads mark the location of emerging ‘daughter’ vesicles. In the hepatocyte, a vesicle

containing both ASOR (red) and Tfn (green) moves to the right and at 30 seconds begins to undergo fission, resulting in a separate daughter

vesicle by 42 seconds. In the in vitro example, vesicles were bound to microtubules (MTs) (red filaments) and labeled for endocytic ASOR

(red) as well as the transferrin receptor (TfR) (green), and ATP was added to initiate motility and fission at 0 seconds. Fission occurred at

21 seconds, resulting in a separate daughter vesicle by 30 seconds. D) Vesicles were assessed for the degree of aggregation in the

presence or absence ofMTs. A rat liver was allowed to endocytose Alexa-488 ASOR (green), and a second liver was allowed to endocytose

Texas red ASOR (red), and the green and red livers were homogenized together and endocytic vesicleswere purified according to protocol.

Most vesicles contained either red or green fluorescence (arrows), indicating that vesicles did not aggregate, although occasional overlap

was seen (arrowhead). The lack of aggregation is better seen when vesicles are imaged in the absence of microtubule (right). Scale bars,

5 mm, except in C, 2 mm.

836 Traffic 2008; 9: 833–847

Murray et al.

obtained in previous studies that used the same procedure

to purify endocytic vesicles. In those studies, 25% of the

ASOR-containing vesicles were motile and 13% of the

motile vesicles underwent fission [(20); n ¼ 832]. A sec-

ond endocytic marker, fluorescent dextran, was injected

into the rat hepatic portal vein instead of fluorescent

ASOR, and vesicles were isolated by the same procedure.

Dextran is a pinocytotic fluid-phasemarker frequently used

to track intracellular vesicles (39,40). In vitromotility assays

were performed, and the level of motility and fission of

dextran-containing vesicles was measured. It was found

that dextran-containing vesicles had higher motility than

ASOR-containing vesicles (58.2%, n ¼ 531, p � 0.001

versus 21% for ASOR) but significantly less fission of the

dextran fluorescence (8.4%, compared with 14% for

ASOR, p � 0.001; Figure 2). This indicates that the level

of motility and fission is different for different vesicle

populations, despite the fact that the vesicles were purified

by the same procedure. Interestingly, the dextran-containing

vesicles had higher motility than the ASOR-containing

vesicles, yet fission was significantly lower. This demon-

strates that fission and motility are regulated independ-

ently. Currently, it is not clear why dextran-containing

vesicles would have such high levels of motility, and we do

not know how these activities affect endocytic processing.

As mentioned above, the endocytic vesicle preparation

contains many different vesicles (11), and, when used as

an endocytic ligand, a subset of the vesicles will contain

ASOR. We had previously determined that many of the

vesicles within the preparation contain the transmembrane

protein, transferrin receptor, as well as the bile acid trans-

porter, ntcp (26). To determine if vesicles containing either

of these proteins had similar levels of motility and fission

as that of the ASOR-containing vesicles, motility assays

were performed for vesicles that were labeled by im-

munofluorescence for either transferrin receptor or ntcp.

Movies resulting from these experiments were analyzed

for vesicles that contained either ntcp or transferrin

receptor but not ASOR. Forty-one per cent of vesicles that

contained ntcp were motile, yet no fissions were detected

(n ¼ 861). Thirty-two per cent of the vesicles that con-

tained transferrin receptor were motile (n ¼ 322), and only

3.8% of these underwent fission (Figure 2). These data

indicate that some vesicles within the preparation have

a low amount of fission, despite having high motility.

Approximately 20% of the vesicles that contain either ntcp

or transferrin receptor also contain ASOR (data not

shown). When these vesicles were analyzed, it was found

that 29.1% of the vesicles that contained ASOR and ntcp

were motile (n ¼ 316) and 26.1% underwent fission of the

ntcp protein (Figure 2). Twenty point eight per cent of the

vesicles that contained transferrin receptor and ASOR

were motile, and 22.2% underwent fission of the trans-

ferrin receptor (n ¼ 303; Figure 2). In both cases, the

presence of the endocytic ligand corresponded with

increased fission and slightly reduced motility. When

vesicles containing endocytic dextran instead of endocytic

ASOR were used for similar studies, it was found that

ntcp, transferrin receptor and several other proteins failed

to show significant colocalization with the fluorescent

dextran in vitro, precluding the generation of additional

data (not shown). Overall, however, these experiments

demonstrated that different vesicles within the same

preparation have different levels of motility and fission

and that vesicles containing ASOR as well as other

proteins have sufficient fission to allow an analysis of the

segregation of ASOR.

Inhibition of PI3-kinase decreases fission but not

motility of vesicles that contain ASOR and ntcp

To confirm that fission and motility can be regulated

independently, we measured these activities following

preincubation with the phosphatidylinositol 3-kinase (PI3-

kinase) inhibitor, LY294002.Many reports have shown that

PI3-kinase inhibition reduces protein recycling and sorting

Figure 2: The level of motility and fission is different for

different populations of endocytic vesicles; fission is low for

vesicles that contain ntcp or transferrin receptor (TfR) but

lack ASOR. Motility and fission were measured in the in vitro

motility assay for vesicles that contained ASOR [n (number of

vesicles) ¼ 67, e (number of experiments) ¼ 5], dextran (n ¼ 531,

e ¼ 10), ntcp but no ASOR (n ¼ 861, e ¼ 7), transferrin receptor

(TfR) but no ASOR (n ¼ 322, e ¼ 4), ntcp and ASOR (n ¼ 316,

e ¼ 22), TfR and ASOR (n ¼ 303, e ¼ 13), and ntcp and ASOR

(n ¼ 212, e ¼ 15) after pretreatment with 50 mM LY294002, as

indicated. Motility was scored as the per cent of microtubule-

bound vesicles that underwent continuous directed movement

(y-axis). Fission was scored as the per cent of moving vesicles that

underwent fission (y-axis). Assays were performed using different

aliquots of the same preparations of endocytic vesicles, which

were purified following endocytic uptake of fluorescent ASOR into

rat liver. In some cases, fluorescent dextran was substituted for

ASOR (as indicated). Different populations of vesicles within the

preparation are indicated on the x-axis. These were identified

using fluorescent antibodies and fluorescent endocytic ASOR,

which labeled subsets of the vesicles within the preparation.

Traffic 2008; 9: 833–847 837

Endocytic Segregation In Vitro

(41–43), and we therefore examined whether the fission of

endocytic vesicles, which may participate in protein sorting

and recycling, may also be affected. PI3-kinase activity has

also been shown to be important for the formation of

phospholipids that allow the attachment of motor proteins

to vesicles (10,44). However, in these studies, LY294002

was added after vesicles had attached to microtubules,

and the PI3-kinase activity might not be limiting for the

motility of pre-attached vesicles.

The motility and fission of microtubule-bound vesicles

containing ASOR and ntcp were quantified following pre-

incubation with 50 mM LY294002 in the motility chamber.

As can be seen in Figure 2 (rightmost bars), 34.4% of the

inhibitor-treated vesicles moved along microtubules (n ¼212) compared with 29.1% of untreated vesicles (n ¼ 316),

suggesting that PI3-kinase inhibition has minimal effect

on the motility of vesicles that contain ASOR and ntcp

(p ¼ 0.2). However, fission was reduced from 26.1 to 9.6%

(p ¼ 0.01), indicating that PI3-kinase regulates the level of

vesicle fission without affecting motility.

Segregation of ASOR from ntcp

Previous studies have shown that the bile acid transporter,

ntcp, resides on the plasma membrane as well as intra-

cellularly (25,45), and recently, we have shown that

a fraction of this protein colocalizes with early endocytic

ASOR but not late endocytic ASOR (26). Fission of ntcp

away from ASOR in early endocytic vesicles represents

a potential means by which ntcp is depleted from the late

endosomes. This was examined in this study bymeasuring

the degree to which ASOR and ntcp segregate during

fission along microtubules. To obtain this data, early

endocytic ASOR-containing vesicles were bound to micro-

tubules in vitro and the presence of ntcp was detected on

the vesicles by immunofluorescence. ATP was added to

the vesicles during multi-channel, time-lapse fluorescence

microscopy experiments. Subsequently, the time-lapse

movies were analyzed for the presence of vesicles that

contain both ntcp and ASOR, and this population was

analyzed for fission. The amount of ASOR and ntcp segre-

gating to each daughter vesicle was determined by mea-

suring the amount of fluorescence for each fission. These

data are tabulated (Supplementary Tables 1, 2 and 6) and

displayed as a correlation of the two proteins (see below).

Figure 3 is a cartoon that indicates how a correlation plot

can be used to view protein segregation. On the left, the

figure depicts a vesicle undergoing fission into two daugh-

ter vesicles, and on the right, the per cent of protein

recovered in each daughter vesicles is plotted as a corre-

lation. Four hypothetical examples demonstrate different

distributions of red and green proteins into daughter

vesicles, while yellow indicates overlap of red and green

in the same vesicle. In the first example, red and green

proteins divide equally between the two daughter vesicles

and the proteins do not segregate. The correlation plot

shows that each daughter vesicle contains approximately

50% of the red and green proteins. In the second example,

the proteins divide unequally into the daughter vesicles but

again there is no segregation. One vesicle contains a large

percentage of both red and green proteins, while the other

vesicle contains a small percentage of both proteins. In this

case, the daughter vesicles reside in the upper right and

lower left quadrants of the correlation plot. In the third

example, the proteins divide unequally into the daughter

vesicles and partial segregation is achieved. One daughter

vesicle contains a large percentage of red protein along

with a small percentage of green protein, while the other

daughter contains a small percentage of red protein along

with a large percentage of green protein. These vesicles

reside in the upper left and lower right quadrants of the plot.

In the final example, complete segregation is achieved, and

one vesicle contains 100% of the red protein and 0% of the

green protein, and the other vesicle contains 0% of the red

protein and 100% of the green protein. These vesicles

reside in the extreme upper left and lower right of the plot.

The correlation plot therefore indicates whether proteins

segregate from each other during fission, and all fissions

are displayed so that the scatter in the data is revealed. The

percentage of vesicles that are found in each quadrant can

be included as an additional measure of the segregation of

the two proteins as is frequently performed for flow

cytometry analysis (46). Proteins that segregate will be

negatively correlated, localizing to the upper left and lower

right quadrants, while proteins that do not segregate will

be positively correlated, localizing to the upper right and

lower left quadrants. Because both daughter vesicles are

included, the graph is rotationally symmetric (each vesicle

is related to the other daughter of the pair by the formula:

100% � co-ordinate value). Also note that the correlation,

plotted as a percentage (as in Figure 3), does not provide

information on the total amount of fluorescence.

Figure 4A presents the correlation plot for vesicle fissions

that contain ntcp and ASOR. Overall, the vesicles cluster to

the upper left and lower right quadrants of the plot,

indicating that the proteins are segregating. The degree

of segregation can be represented by the average values

of the lower left and lower right quadrants. Vesicles in

these lower quadrants contain the smaller portion of

ASOR. They also contain some proportion of ntcp. If

protein divided equally likely into any portion and if the

proteins distributed independently from one another, then

the average value of the lower quadrants would be 25%

for ASOR and 50% for ntcp. Twenty-five per cent is the

middle value for the lower half of a population, and 50%

is the middle value for a total population. However, in

the actual correlation plot, these quadrants contained

11.3 � 1.6% (mean � SEM) of the ASOR and 72.9 �3.0% of the ntcp, as indicated by the triangle and dotted

lines of Figure 4A. The scatter of the data indicates that

the per cent of protein distributing to daughter vesicles

is variable. In addition, there are many vesicles that

838 Traffic 2008; 9: 833–847

Murray et al.

completely lack ASOR and some that completely lack ntcp,

and therefore, some fission events achieve complete

segregation of the proteins. It should also be noted that

if the observed fission was actually the result of independ-

ent movement of overlapped ‘pure’ vesicles (i.e. vesicles

that contained only ASOR or only ntcp), then ‘mixed’

daughter vesicles (containing both ntcp and ASOR) should

rarely be observed. Instead, about 60% of all daughter

vesicles were mixed. In summary, these results suggest

that intracellular fission produces daughter vesicles con-

taining a broad distribution of protein, with many vesicles

(32 of 130 or 25%) completely devoid of ASOR, a marker

that is targeted for degradation within lysosomes.

Segregation of ASOR from transferrin receptor

Several other protein markers were selected for study

in order to determine whether segregation during fis-

sion is dependent on the identity of the protein and

whether proteins that are targeted for different cellular

destinations undergo greater segregation than those

that are targeted for the same destination. Figure 4B

presents a correlation plot for vesicles containing

transferrin receptor and ASOR. These experiments were

performed identically to the experiments containing ntcp

except that antibodies to transferrin receptor were

included to identify this protein. Figure 4B indicates

that the transferrin receptor also segregates away from

Figure 3: Graphing the distribution of protein from vesicle fission as a correlation. A cartoon indicates several examples of how two

proteins may distribute to different vesicles during fission and how this can be plotted as a correlation. A mother vesicle containing red and

green proteins is shown on the left bound to a microtubule (red filament); yellow indicates overlap of the red and green proteins. The

mother vesicle undergoes fission (arrow) into two daughter vesicles, and the fraction of red and green proteins distributing to each

daughter is plotted as a percentage as two dots. In the first example, protein distributes approximately equally to the daughter vesicles, and

the daughter vesicles appear centrally localized on the correlation plot. In the second example, protein distributes unequally to the

daughters, but red and green proteins do not segregate. These daughters localize to the lower left and upper right quadrants of the plot.

In the third example, protein distributes unequally to the daughters, and red and green proteins partially segregate. Vesicles from such

fissions localize to the upper left and lower right quadrants of the plot. In the final example, the proteins segregate completely, and

daughter vesicles align at the extreme upper left (0 and 100%) and lower right (100 and 0%) portions of the plot. Because both daughter

vesicles are included, the graph is rotationally symmetric (each vesicle is related to the other daughter of the pair by the formula:

100% � co-ordinate value).

Traffic 2008; 9: 833–847 839

Endocytic Segregation In Vitro

Figure 4: Segregation of ASOR from ntcp and transferrin receptor during microtubule-based motility in vitro. Vesicles were

purified from rat liver that contained fluorescent endocytic ASOR as well as subpopulations of unlabeled vesicles as described in Figure 2.

These were bound to microtubules and labeled for ntcp and transferrin receptor by immunofluorescence. Motility assays were performed

and subsequently analyzed for vesicles that have undergone fission and contain ASOR and ntcp or transferrin receptor. The amount of

protein present on each endocytic vesicle was measured by fluorescence intensity. The distribution of ASOR was plotted as a correlation

versus ntcp or transferrin receptor in the manner outlined in the cartoon of Figure 3. Each point represents a single daughter vesicle after it

has undergone fission. In (A) and (B), the per cent of protein segregating to daughter vesicles is presented, and the graph is rotationally

symmetric. In (C) and (D), the total amount of fluorescence is presented. Fissions showing identical percentages were offset by 0.5%

along the x-axis for visualization. A) ASOR and ntcp predominantly segregated to opposite vesicles during fission. The lower quadrants

contained an average of 11.3 � 1.6% (SEM) of the ASOR and 72.9 � 3.0% of the ntcp, as indicated by the triangle and dotted line.

B) ASOR and transferrin receptor segregated to opposite vesicles during fission, although the trend was not as strong as for ASOR and

ntcp. The lower quadrants contained an average of 10.9 � 1.7% of the ASOR and 61.0 � 3.2% of transferrin receptor as indicated by the

triangle and dotted line. C) A plot of total fluorescence (rather than per cent) of ASOR versus ntcp for the same vesicles that were used in

(A). This does not reveal a negative correlation, which indicates that the negative correlation of (A) is a result of fission rather than the

amount of protein on the vesicles per se. D) A plot of the total fluorescence of ASOR versus transferrin receptor for the same vesicles that

were used in (B). Fluorescence is in arbitrary units for all probes. UL, UR, LL and LR are abbreviations for upper left, upper right, lower left

and lower right, and the adjacent number indicates the percentage of vesicles that are found in the quadrant.

840 Traffic 2008; 9: 833–847

Murray et al.

ASOR during fission. The lower right and lower left

quadrants contained 10.9 � 1.7% of the ASOR and

61 � 3.2% of transferrin receptor, as indicated by the

triangle and dotted lines. There is also a similar degree of

scatter and a similar amount of vesicles that completely

lack ASOR (30%).

Lack of correlation between total fluorescence

in daughter vesicles

It could be argued that the degree of protein segregation

that we have attributed to fission is in reality a reflection of

the degree to which the proteins are present in vesicles

overall. That is, perhaps those vesicles that contain a low

amount of ASOR also contain a high amount of ntcp,

independent of whether or not these vesicles have under-

gone fission. We tested this hypothesis by plotting a cor-

relation for the total amount of fluorescence, rather than

the per cent, that is present on the vesicles of the previous

two data sets. Figure 4C,D shows that there is no strong

overall trend for either ntcp or transferrin receptor with

respect to ASOR, that is, vesicles containing either a high

or a low amount of ASOR can contain a high or low amount

of ntcp or transferrin receptor. It is only when the vesicles

are paired because of fission (Figure 4A,B) that we see

a low amount of ASOR distributing with a high amount of

ntcp. Mean values were used to divide the total fluores-

cence data into quadrants in the manner of Figure 4A,B,

and these did not reveal any unusual distributions

of protein. Eighty-three per cent and 68% of the vesicles

were found in the negatively correlated (upper left and

lower right) quadrants in Figure 4A,B, while 47 and 55% of

the vesicles were found in the negatively correlated

quadrants of Figure 4C,B.

Segregation of transferrin receptor from ntcp, ASOR

from ASGPR and ASOR from EGFR

Figure 5A presents a correlation plot for vesicles that con-

tain transferrin receptor and ntcp. Unlike ASOR, transferrin

Figure 5: Segregation of Transferrin receptor from ntcp,

ASOR fromASGPR and ASOR from EGFR during microtubule-

based motility in vitro. Vesicles were purified from rat liver that

contained fluorescent endocytic ASOR as well as subpopulations

of unlabeled vesicles as described in Figure 2. These were used

for in vitromotility assays, and the per cent of protein segregating

to individual vesicles was plotted as correlations as in Figure 4.

A) Transferrin receptor and ntcp remain together (i.e. do not seg-

regate) during fission. The lower left and lower right quadrants

(vesicles containing the lower portion of transferrin receptor)

contained on average 30 � 4.6% of the transferrin receptor and

27 � 4.8% of ntcp. B) ASOR and ASGPR partially segregate

during fission. The lower quadrants contained on average

11 � 3.8% of the ASOR and 46 � 6.8% of the ASGPR. C) ASOR

and EGFR partially segregate during fission. The lower quadrants

contained on average 18 � 3.7% of the ASOR and 40 � 6.3% of

the EGFR. UL, UR, LL and LR are abbreviations for upper left,

upper right, lower left and lower right, and the adjacent number

indicates the percentage of vesicles that are found in the quadrant.

The triangles and dotted lines indicate average values for the lower

quadrants.

Traffic 2008; 9: 833–847 841

Endocytic Segregation In Vitro

receptor did not segregate from ntcp during fission.

Instead, 86% of the daughter vesicles were contained in

the upper right and lower left (positively correlated) quad-

rants of the plot. The lower left and lower right quadrants

(vesicles containing the lower portion of transferrin recep-

tor) contained on average 30 � 4.6% of the transferrin

receptor and 27 � 4.8% of ntcp, indicating that vesicles

with a low amount of transferrin receptor also contained

a low amount of ntcp on average. Few vesicles (4 of 42

or 10%) completely lacked transferrin receptor. Cumula-

tively, these data indicate that two basolateral, plasma

membrane proteins, ntcp and transferrin receptor, are able

to efficiently segregate from the endocytic ligand, ASOR,

during microtubule-based fission while they are unable to

segregate from each other.

ASGPR, the receptor for ASOR, was chosen as a fourth

marker for the analysis of protein segregation. ASGPR–

ASOR defines a classical pathway whereby receptor binds

to ligand at the cell surface, releases ligand intracellularly

and the ligand is targeted to lysosomes where it is

degraded, while the receptor is recycled back to the cell

surface (47,48). Further studies showed that approxi-

mately half of the ligand dissociates from the receptor

rapidly (t½ � 2.5 min) following endocytic uptake, while

the other half dissociates slowly (t½ � 50 min). Therefore,

for reasons that are still not understood, a significant

portion of ASOR remains bound to ASGPR during endo-

cytic processing and vesicular traffic (49,50).

We used the in vitro assay to address whether ASOR that

is located within endocytic vesicles after 5 min of uptake

undergoes efficient segregation from ASGPR during

microtubule-based fission. Figure 5B shows that segrega-

tion between ASOR and ASGPR was not as efficient as

the segregation of ASOR from either ntcp or transferrin

receptor. On average, 11 � 3.8% of ASOR and 46� 6.8%

of ASGPR were present in the lower quadrants of the

correlation plot. A similar level of segregation of ASGPR

from ASOR was also observed in a previous study (20).

This level of segregation was intermediate between the

segregation of ASOR from ntcp (proteins that strongly

segregated) and transferrin receptor from ntcp (proteins

that did not segregate). The percentage of vesicles in the

negatively correlated upper left and lower right quadrants

totaled 83, 35 and 14% for the segregation of ASOR from

ntcp, ASOR from ASGPR and transferrin receptor from

ntcp, respectively. Interestingly despite moderate overall

segregation, 29% of vesicles completely lacked ASOR as

a result of fission of vesicles containing ASGPR and ASOR.

These studies suggest that ASOR is not completely free to

segregate from ASGPR within the endocytic vesicles and

that the slowly dissociating [‘state 1’ (48)] receptors are

present within endocytic vesicles that can bind to and

move along microtubules.

The EGFR was chosen as a fifth marker for the study of

endocytic segregation as it is an integral membrane recep-

tor that follows a different intracellular trafficking pathway

than either transferrin receptor or ASGPR. Whereas the

transferrin receptor and ASGPR are recycled to the plasma

membrane following endocytosis, the EGFR is taken up by

clathrin-mediated endocytosis and transported to the inner

membranes of multivesicular bodies where a significant

portion of the receptormoves to lysosomes and is degraded

(23,24,51). Figure 5C shows that the EGFR does not sort

away from the degraded ligand, ASOR, during microtubule-

based fission. On average, 18 � 3.7% of the ASOR and

40 � 6.3% of the EGFR were present in the lower quad-

rants of the correlation plot, and only 40% of the vesicles

were present in the negatively correlated upper left and

lower right quadrants of Figure 5C. These studies suggest

that the different endocytic sorting pathways of EGFR and

transferrin receptor are reflected in their different sorting

behavior with respect to ASOR.

Summary of the sorting behavior of the proteins

Table 1 summarizes the sorting of the different combin-

ations of proteins that were studied in this report. Each

fission is the division of a single vesicle into two vesicles,

and therefore, the amount of protein sorting to each

vesicle was plotted as per cent of total (Figures 4A,B and

5A–C). Strong segregation would be indicated by a low

percentage of one protein sorting into the same vesicles

that contain a high percentage of the other protein. This

is shown in Table 1 as the ‘average per cent of protein

Table 1: Summary of the degree of protein segregation during

fission

Protein pair

analyzed

Average per cent

protein recovered

in one daughter

vesiclea

Fold

purificationbnc Per cent of

fissions with

protein

segregationd

ASOR 11.3 � 1.6 6.5 65 83

Ntcp 72.9 � 3.0

ASOR 10.9 � 1.7 5.6 72 68

TfR 61.0 � 3.2

ASOR 18.4 � 3.7 2.2 15 40

EGFR 39.7 � 6.3

ASOR 11.2 � 3.8 4.1 17 35

ASGPR 45.8 � 6.8

TfR 29.9 � 4.6 0.9 21 14

Ntcp 27.4 � 4.8

aThis is the average per cent protein � SEM in the lower left and

lower right quadrants of Figures 4A,B and 5A–C. These are the

daughter vesicles from each fission that contain the smaller

portion of the first listed protein.bThis is the purification of the second listed protein from the first in

these vesicles, compared with the pre-fission vesicle, obtained by

dividing the percentages in the previous column.cThe number of fissions.dThis is the per cent of vesicles located within the negatively

correlated quadrants of Figures 4A,B and 5A–C. In these fissions,

the majority of one protein was found in the vesicle containing the

minority of the other protein.

842 Traffic 2008; 9: 833–847

Murray et al.

recovered in one daughter vesicle’ and corresponds to the

average per cent of protein in the lower left and lower right

quadrants of the correlation plots. About 11.3% of the

ASOR and 72.9% of the ntcp were found in the same

vesicles, indicating that a single round of fission produced

a 6.5-fold purification of ntcp (72.9/11.3%) on average.

Table 1 ranks the proteins according to their tendency to

segregate during fission. Ntcp segregated strongly from

ASOR, transferrin receptor segregated slightly less

strongly, and EGFR and ASGPR had moderate segregation

from ASOR. Transferrin receptor, however, did not segre-

gate from ntcp. This can be seen in the column that lists

the ‘per cent fissions with protein segregation’ and corres-

ponds to the per cent of vesicles that are contained in the

negatively correlated, upper left and lower right quadrants.

The degree of segregation was significantly different for all

protein markers with p-values of 0.01, <0.005 and <0.005

when comparing the per cent of ntcp segregating from

ASOR to the per cent of transferrin receptor, EGFR and

ASGPR segregating from ASOR. These data indicate that

proteins have different degrees of segregation during

fission, and it suggests that microtubule-based fission

events are participating in the segregation of protein.

The degree of segregation does not correlate with

vesicle fluorescence intensity or size

A population of ‘highly sorted’ daughter vesicles was

analyzed for their fluorescence intensity and motility.

Daughter vesicles arising from fission were considered

highly sorted if they contained at least 80% of a single

marker protein and less than 20% of the ‘opposing’ marker

proteins. For ASOR, both ntcp and transferrin receptor

were considered opposing marker proteins, while for ntcp

and transferrin receptor, ASOR was considered the oppos-

ing marker protein (Table 2). The highly sorted vesicles

were brighter on average than the remaining vesicles,

indicating that protein segregation did not occur into dim

vesicles. The highly sorted vesicles also had equal or

greater standard deviation of fluorescence, indicating that

protein did not segregate into vesicles of uniform intensity.

Overall, this analysis did not reveal any correlation

between fluorescence intensity and the degree of protein

sorting. Furthermore, the total fluorescence correlation

plots of Figure 4C,D showed that fluorescence intensities

were dispersed. These results are consistent with the

concept that protein segregates to vesicles that have

a broad distribution in size and intensity (52).

The transmembrane proteins, ntcp and transferrin

receptor, were highly motile during fission

Fissions were observed as the division of a single vesicle

into two daughter vesicles. Typically, one of the daughter

vesicles was motile, while the other remained stationary,

although in some fissions (6%) both daughters were

motile (Supplementary Tables). A review of the data

shows that some proteins were less abundant in the

moving vesicles than others. For instance, on average,

66% of the daughter vesicles in the lower left and lower

right quadrants of Figures 4A,B and 5A–C were motile.

These are the daughter vesicles that contained the smaller

amount of ASOR from the fission. The trend is more

apparent when looking at fissions that strongly segregated

protein. Seventy-two per cent of daughter vesicles that

contained either ntcp or transferrin receptor but were

completely depleted of ASOR after fission were motile

(n ¼ 60, culled from Tables S1–S6). However, only 38% of

the vesicles that contained ASOR but were completely

depleted of ntcp were motile (n ¼ 16, p ¼ 0.01), and 20%

of vesicles that contained ASOR but were completely

depleted of transferrin receptor were motile (n ¼ 11,

p < 0.001). This is also seen in the analysis of ‘highly

sorted’ vesicles of Table 2. Seventy per cent and 86% of

the highly sorted ntcp- and transferrin receptor-containing

vesicles, respectively, were motile compared with 41%

of the highly sorted ASOR-containing vesicles (n ¼ 27,

p ¼ 0.03 and p < 0.001, respectively, compared with highly

sorted ASOR-containing vesicles). These data show that

fissions were frequently the result of the transmembrane

proteins, ntcp and transferrin receptor, moving away from the

ligand, ASOR, that is located on the lumenal side of the

vesicle. This is consistent with the concept of ‘geometric’

protein sorting inwhich transmembrane proteins are believed

to be concentrated into tubules that bud or pull away from

the endocytic lumen, resulting in protein segregation (8,53).

Discussion

This study demonstrates that microtubule-based motility

results in segregation of endocytic protein and that the

Table 2: Properties of the highly sorted vesiclesa

Protein Highly sorted

vesicles

Remaining

vesicles

ASOR

Mean intensity 337.7 244.5

SD 305.8 355.3

Per cent moving 41 52

n 27 172

Ntcp

Mean intensity 399.5 301.3

SD 335.3 347.3

Per cent moving 70 54

n 20 90

Transferrin receptor

Mean intensity 717.9 477.8

SD 585.9 478.0

Per cent moving 86 50

n 14 112

aThese are a subset of fissions where proteins strongly sorted

from each other. They contained at least 80% of the listed protein

and less than 20% of the ‘opposite’ protein. For ASOR, the

opposite protein was considered as both ntcp and TfR, and for

ntcp and TfR, the opposite protein was considered as ASOR.

Traffic 2008; 9: 833–847 843

Endocytic Segregation In Vitro

degree of segregation is specific to the particular protein.

In living cells, internalized ligands were seen to undergo

multiple microtubule-based vesicle fissions, resulting in

apparent segregation of protein (Figure 1). However, vary-

ing background fluorescence, photobleaching and the

complex, three-dimensional motion of the vesicles pre-

vented accurate quantification of protein fluorescence.

Instead, we used endocytic vesicles prepared from rat

liver to recapitulate microtubule-based traffic within in vitro

microscopy assays. This allowed direct quantification of

protein with fluorescent antibodies and fluorescent-tagged

ligands. Previous reports have shown that movement of

the vesicles is dependent on specific sets of motor

proteins and that the inhibition of motor proteins blocks

fission (11,20,26,33). Fission resulted from splitting and

movement of at least one daughter vesicle along the

microtubule in every case (Supplementary Tables).

It is possible that fission under these conditions is depend-

ent only on the activity of motor proteins. However, we

found that fission is regulated differently than vesicle

motility. For instance, vesicles containing transferrin recep-

tor and ASOR had decreased motility but increased fission

compared with vesicles containing transferrin receptor but

no ASOR. Also, PI3-kinase inhibition reduced fission but

not motility (Figure 2). This suggests that fission on micro-

tubules results from the co-ordinated activity of motor pro-

teins with other cellular machineries. These may include

proteins that have been shown to be involved in vesicle

budding and protein sorting such as coatomers, adaptors

and ADP ribosylation factor 1 (54). It should be emphasized

that our studies were performed without addition of exo-

genous proteins and that soluble material is removed

during the vesicle purification and microscopy assays.

The vesicles themselves had all the machinery required

to produce motility, fission and protein segregation.

Analyses of protein levels on vesicles undergoing fission

suggest that proteins segregate during fission only when

they are destined for different cellular compartments. Ntcp

strongly segregated away from ASOR (Table 1), and this

was achieved because ASOR sorted asymmetrically, leav-

ing many ntcp vesicles completely devoid of ASOR (Fig-

ure 4A). The transferrin receptor showed a similar but

somewhat weaker segregation from ASOR (Figure 4B;

Table 1), whereas ntcp and transferrin receptor, proteins

that are both recycled to the plasma membrane, remained

together during fission (Figure 5A). ASOR showed moder-

ate segregation from its receptor, ASGPR. Although the

receptor is recycled and the ligand is degraded, a significant

portion of ASOR remains bound to receptor following

endocytosis and only dissociates slowly (49). Similar slowly

dissociating pools of ligand are seen for many if not most

receptor–ligand pairs, and a full understanding of this

phenomenon remains to be elucidated. EGFR, an integral

membrane protein that is targeted for degradation (23), also

failed to segregate efficiently from ASOR. It is interesting to

consider that neither ASGPR nor EGFR segregates effi-

ciently from ASOR in vitro. However, within cells, ASGPR is

recycled to the plasmamembrane, while EGFR is degraded.

Analysis of the data indicates that, although their overall

segregation from ASOR is similar, more ‘pure’ ASGPR

vesicles are generated than pure EGFR vesicles (Figure

5B,C). This suggests that the generation of pure vesicles is

an important step for protein segregation, and we speculate

that these vesicles may be directly targeted to the recycling

compartment and plasma membrane.

The geometric model of protein segregation proposes that

vesicles enriched in transmembrane receptors are physi-

cally pulled away from lumenal protein (53). Agreeing with

this, we found that fissions frequently produced a motile

daughter vesicle that was enriched in transmembrane

protein (Table 2). A recent study that tracked the move-

ment of endocytic vesicles within cells showed that

ligands can pre-sort to dynamic and static endosome

populations and that the dynamic population is more

rapidly processed for degradation (6). We also observed

populations of vesicles with different rates of motility.

However, in our studies, vesicles containing proteins that

are destined for recycling (ntcp and transferrin receptor)

had similar or higher motility than vesicles containing

proteins or molecules that are destined for degradation

(ASOR and dextran; Figure 2). Many factors can regulate

motility (5,26,55), and additional studies will be needed to

understand this regulation. The recycling of membrane

transporters such as GLUT4 or Naþ/Hþ exchanger (42,43)

has been found to be dependent on PI3-kinase activity, and

in this study, it is shown that PI3-kinase inhibition reduces

fissions by more than half without affecting vesicle motil-

ity. Studies from our laboratory have found that inhibition

of the microtubule-based minus-ended motor KIFC1 de-

creased fission (33) and that rab4 may have a role (55)

in regulation of the fission machinery. Activation of the

molecular motors by protein kinases (56,57) and concom-

itant involvement of the Rab proteins represent potential

mediators of membrane fission and vesicle processing.

Materials and Methods

Chemicals and reagentsCommercial antibodies used were transferrin receptor (number 13-6800;

Zymed laboratories) and EGFR (number sc-03; Santa Cruz Biotechnology).

In-house-generated antibodies used were ASGPR, to the C-terminal

cytoplasmic tail (20), ntcp, to amino acids 337–350 at the cytoplasmic

C-terminus (26). Primary antibodies were used at 0.33–20 mg/mL. Fluores-

cent secondary antibodies were from Jackson ImmunoResearch. ASOR

was prepared from human orosomucoid (Sigma-Aldrich) by acid hydrolysis

(58) and labeled with Texas red sulfonyl chloride or Alexa-488 (Invitrogen

Corporation) as described (32). Some ASOR was labeled with 125I by

a chloramine-T method to a specific activity of 10 000–15 000 c.p.m./ng

(59). Amino dextran (Cat. no. D1861; Invitrogen Corporation) was labeled

with Texas red sulfonyl chloride. Labeled and unlabeled tubulin was from

Cytoskeleton. All other reagents were from Sigma-Aldrich.

Fluorescent ASOR-containing endocytic vesicles were prepared from rat

liver as previously described (11,20,26,32,60,61). For some experiments,

the portal veins of two livers were separately injected with either Texas red

844 Traffic 2008; 9: 833–847

Murray et al.

or Alexa-488 ASOR, and these were homogenized together and vesicles

were isolated according to protocol. Taxol-stabilized fluorescent micro-

tubules were polymerized from rhodamine–tubulin as previously described

(11). Assay of vesicle motility on microtubules was as described (20,32,60).

Antibodies were screened for their appropriateness in immunofluores-

cence assays by Western blot and whole cell immunofluorescence (37).

For a given day’s experiment, 10–15 chambers with microtubule-bound,

immunofluorescence-stained endosomes were assembled and stored on

ice, and the chambers were sequentially placed on a microscope stage

maintained at 378C. Time-lapse multi-fluorescence channel microscopy

was then initiated, and 50 mM ATP was added to induce movement and

fission of the endosomes.

Immunofluorescence analysis in vitroDetection of protein on unfixed endocytic vesicles bymonoclonal or affinity-

purified polyclonal antibody followed by fluorescence secondary antibody

was performed as described (60). Wide-field imaging was performed at

378C using a 60�, 1.4 numerical aperture Olympus objective on an

Olympus 1X71 inverted microscope equipped for multi-channel fluores-

cence with a Lambda DG-4 excitation source and Lambda 10-2 emission

filter wheel (Sutter Instruments). Data were collected through a CoolSnap

HQ cooled charge-coupled device camera (Photometrics, Roper Scientific)

using METAMORPH software (Molecular Devices). Time-lapse movies were

captured in 1–3 channels (Cy2, Cy3 and Cy5). Some images were captured

on a BioRad Radiance confocal microscope in the Analytical Imaging Facility

(Albert Einstein College of Medicine). Movies were analyzed using IMAGEJ

software (National Institutes of Health public domain; http://rsb.info.nih.-

gov/ij/) and ADOBE PHOTOSHOP v 6.0 (Adobe Systems Inc.). Protein signal was

measured as the signal intensity minus background multiplied by the area.

Background intensity was measured and averaged from three neighboring

regions. To adjust for different imaging conditions (e.g. exposure time and

excitation intensity), fluorescence was normalized so that each set of

experiments gave equal median vesicle fluorescence for a given protein.

For total fluorescence correlation plots, 10 units (in arbitrary units) were

added to each vesicle to allow plotting on a logarithmic scale. The p-values

for correlation coefficients were from Student’s t-test. The Supplementary

Tables present all the data from different sets of experiments that were

performed either with different combinations of antibodies or after pre-

incubation with 50 mM LY294002. In the correlation plots, fissions were

culled from all experimental sets where the listed proteins were used,

regardless of the presence of other proteins.

ASOR uptake in cultured rat hepatocytesRat hepatocytes were isolated by collagenase perfusion as described (55),

attached to coverslip-bottomed dishes (MatTek Corporation) that were

coated with 0.5 mg/mLMatrigel (BD Biosciences) and cultured overnight in

Hepatozyme media (Invitrogen Corporation). Dishes were treated with

10 mg/mL Texas red-labeled ASOR and, in some experiments, 20 mg/mL

Alexa-488 transferrin (Invitrogen Corporation) for 5 min, washed and

imaged at 378C with 200-millisecond exposure times. Images were pro-

cessed with a SpotEnhancing filter (Daniel Sage, Biomedical Imaging Group).

For some experiments, cells were cooled to 48C in the presence or absence

of 30 mM nocodazole for 30 min, followed by warming to 378C and exposure

to ASOR as described above in continuous exposure to nocodazole.

Acknowledgments

This study was supported by National Institutes of Health grants DK41918

and DK23026.

Supplementary Materials

Table S1: Per cent of protein, motility and other data for daughter vesicles

resulting from fissions of vesicles containing ASOR and ntcp

Table S2: Data from fissioned vesicles containing ASOR, ntcp and trans-

ferrin receptor

Table S3: Data from fissioned vesicles containing ASOR and transferrin

receptor

Table S4: Data from fissioned vesicles containing ASOR, ASGPR and

transferrin receptor

Table S5: Data from fissioned vesicles containing ASOR, EGFR and

transferrin receptor

Table S6: Data from fissioned vesicles containing ntcp and ASOR, and

experiments performed after incubation with the PI3-kinase inhibitor,

LY294002

Movie S1: Endocytic vesicle movement in a control cell (Figure 1A).

Hepatocytes were allowed to take up fluorescent ASOR for 5 min, washed

and after additional 8 min, time-lapse movies of the endocytic vesicles

were captured at 1 frame per 1.2 seconds for a total of 72 seconds. A

hepatocyte is shown with multiple endocytic vesicles traversing the cell

and undergoing varied morphological changes, such as fusion, fission and

tubulation. Scale bar, 5 mm.

Movie S2: Endocytic vesicle movement in a nocodazole-treated cell

(Figure 1A). Hepatocytes were treated with 30 mM nocodazole to depoly-

merize microtubules and then allowed to take up ASOR, followed by

washing as in Movie S1. Time-lapse images were captured at 1 frame per

3 seconds for a total of 90 seconds. Note the lack of long-distance vesicle

movement. Short-distance, oscillating behavior is still observed. Scale bar,

5 mm.

Movie S3: ASOR and transferrin endocytic vesicle movement in control

cells (Figure 1B). Hepatocytes were allowed to take up both Texas red

ASOR (red) and Alexa-488 transferrin (green) for 5 min, washed and imaged

and displayed as described above but with two fluorescence channels.

Time-lapse images were captured at 1 frame per 1.5 seconds for a total of

90 seconds. Both transferrin and ASOR undergo long-distance movement

with morphological changes. Scale bar, 5 mm.

Movie S4: The corresponding bright-field images of Movie S3. Scale

bar, 5 mm.

Movie S5: Endocytic fission of ASOR and transferrin in a living cell (also

presented in Figure 1C) magnified fromMovie S3. A vesicle containing both

transferrin and ASOR moves from lower left toward the upper right. At

frame 26, fluorescence divides and the resulting daughter vesicle moves to

the left. Scale bar, 2 mm.

Movie S6: Second example of endocytic fission of ASOR and transferrin

magnified fromMovie S3. A vesicle containing ASOR and transferrin moves

from central left toward the lower right and then back again. At frame 21

a green daughter emerges. Scale bar, 2 mm.

Movie S7: Endocytic fission of ASOR and transferrin receptor in vitro (see

Figure 1C for an additional example). Endocytic vesicles containing fluor-

escent ASOR (red dots) were isolated from rat liver and subsequently

bound to microtubules (red) and immunostained for transferrin receptor

(blue dots) or ntcp (green dots) within microscope chambers. ATP was

added to initiate motility. Time-lapse images were captured at 1 frame per

1.5 seconds for a total of 24.5 seconds. A vesicle containing ntcp and

ASOR moves to the lower left and at the same time undergoes a fission

event. Scale bar, 2 mm.

Traffic 2008; 9: 833–847 845

Endocytic Segregation In Vitro

Movie S8: Second example of endocytic fission captured as described in

Movie S7. A vesicle containing ntcp, transferrin receptor and ASOR

undergoes a fission event, and the resulting daughter vesicle moves to

the right. Total time was 19.5 seconds. Scale bar, 5 mm.

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

References

1. Gruenberg J. The endocytic pathway: a mosaic of domains. Nat Rev

Mol Cell Biol 2001;2:721–730.

2. Maxfield FR, McGraw TE. Endocytic recycling. Nat Rev Mol Cell Biol

2004;5:121–132.

3. Wessels E, Simpson JC. Impact of live cell imaging on coated vesicle

research. Semin Cell Dev Biol 2007;18:412–423.

4. Bonifacino JS, Traub LM. Signals for sorting of transmembrane

proteins to endosomes and lysosomes. Annu Rev Biochem 2003;72:

395–447.

5. Rink J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mech-

anism of progression from early to late endosomes. Cell 2005;122:

735–749.

6. Lakadamyali M, Rust MJ, Zhuang X. Ligands for clathrin-mediated

endocytosis are differentially sorted into distinct populations of early

endosomes. Cell 2006;124:997–1009.

7. Matteoni R, Kreis TE. Translocation and clustering of endosomes and

lysosomes depends on microtubules. J Cell Biol 1987;105:1253–1265.

8. Geuze HJ, Slot JW, Strous GJ, Lodish HF, Schwartz AL. Intracellular

site of asialoglycoprotein receptor-ligand uncoupling: double-label

immunoelectron microscopy during receptor-mediated endocytosis.

Cell 1983;32:277–287.

9. Harding C, Levy MA, Stahl P. Morphological analysis of ligand uptake

and processing: the role of multivesicular endosomes and CURL in

receptor-ligand processing. Eur J Cell Biol 1985;36:230–238.

10. Hoepfner S, Severin F, Cabezas A, Habermann B, Runge A, Gillooly D,

Stenmark H, Zerial M. Modulation of receptor recycling and degrada-

tion by the endosomal kinesin KIF16B. Cell 2005;121:437–450.

11. Bananis E, Nath S, Gordon K, Satir P, Stockert RJ, Murray JW, Wolkoff

AW.Microtubule-dependentmovement of late endocytic vesicles in vitro:

requirements for dynein and kinesin. Mol Biol Cell 2004;15:3688–3697.

12. Wolkoff AW, Klausner RD, Ashwell G, Harford J. Intracellular segrega-

tion of asialoglycoproteins and their receptor: a prelysosomal event

subsequent to dissociation of the ligand-receptor complex. J Cell Biol

1984;98:375–381.

13. Breitfeld PP, McKinnon WC, Mostov KE. Effect of nocodazole on

vesicular traffic to the apical and basolateral surfaces of polarized

MDCK cells. J Cell Biol 1990;111:2365–2373.

14. Hunziker W, Male P, Mellman I. Differential microtubule requirements

for transcytosis in MDCK cells. EMBO J 1990;9:3515–3525.

15. Maples CJ, Ruiz WG, Apodaca G. Both microtubules and actin

filaments are required for efficient postendocytotic traffic of the poly-

meric immunoglobulin receptor in polarized Madin-Darby canine kidney

cells. J Biol Chem 1997;272:6741–6751.

16. Koval M, Pagano RE. Lipid recycling between the plasma membrane

and intracellular compartments: transport and metabolism of fluores-

cent sphingomyelin analogues in cultured fibroblasts. J Cell Biol 1989;

108:2169–2181.

17. McGraw TE, Dunn KW, Maxfield FR. Isolation of a temperature-

sensitive variant Chinese hamster ovary cell line with a morphologically

altered endocytic recycling compartment. J Cell Physiol 1993;155:

579–594.

18. Block MR, Glick BS, Wilcox CA, Wieland FT, Rothman JE. Purification

of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.

Proc Natl Acad Sci U S A 1988;85:7852–7856.

19. Spang A, Schekman R. Reconstitution of retrograde transport from the

Golgi to the ER in vitro. J Cell Biol 1998;143:589–599.

20. Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW. Microtubule

and motor-dependent endocytic vesicle sorting in vitro. J Cell Biol

2000;151:179–186.

21. Dunn KW, McGraw TE, Maxfield FR. Iterative fractionation of recycling

receptors from lysosomally destined ligands in an early sorting endo-

some. J Cell Biol 1989;109:3303–3314.

22. Huang T, Wolkoff AW, Stockert RJ. Adaptor heat shock protein

complex formation regulates trafficking of the asialoglycoprotein re-

ceptor. Am J Physiol Gastrointest Liver Physiol 2006;290:G369–G376.

23. Dikic I. Mechanisms controlling EGF receptor endocytosis and degra-

dation. Biochem Soc Trans 2003;31:1178–1181.

24. Renfrew CA, Hubbard AL. Degradation of epidermal growth factor

receptor in rat liver. Membrane topology through the lysosomal

pathway. J Biol Chem 1991;266:21265–21273.

25. Mukhopadhayay S, Ananthanarayanan M, Stieger B, Meier PJ, Suchy

FJ, Anwer MS. cAMP increases liver Naþ-taurocholate cotransport by

translocating transporter to plasma membranes. Am J Physiol 1997;

273:G842–G848.

26. Sarkar S, Bananis E, Nath S, Anwer MS, Wolkoff AW, Murray JW.

PKCzeta is required for microtubule-based motility of vesicles contain-

ing the ntcp transporter. Traffic 2006;7:1078–1091.

27. Marbet P, Rahner C, Stieger B, Landmann L. Quantitative microscopy

reveals 3D organization and kinetics of endocytosis in rat hepatocytes.

Microsc Res Tech 2006;69:693–707.

28. Herman B, Albertini DF. A time-lapse video image intensification

analysis of cytoplasmic organelle movements during endosome trans-

location. J Cell Biol 1984;98:565–576.

29. Goltz JS, Wolkoff AW, Novikoff PM, Stockert RJ, Satir P. A role for

microtubules in sorting endocytic vesicles in rat hepatocytes. Proc Natl

Acad Sci U S A 1992;89:7026–7030.

30. Stockert RJ. The asialoglycoprotein receptor: relationships be-

tween structure, function, and expression. Physiol Rev 1995;75:

591–609.

31. Driskell OJ, Mironov A, Allan VJ, Woodman PG. Dynein is required for

receptor sorting and the morphogenesis of early endosomes. Nat Cell

Biol 2007;9:113–120.

32. Murray JW, Bananis E, Wolkoff AW. Reconstitution of ATP-dependent

movement of endocytic vesicles along microtubules in vitro: an

oscillatory bidirectional process. Mol Biol Cell 2000;11:419–433.

33. Nath S, Bananis E, Sarkar S, Stockert RJ, Sperry AO, Murray JW,

Wolkoff AW. Kif5B and Kifc1 interact and are required for motility and

fission of early endocytic vesicles in mouse liver. Mol Biol Cell 2007;18:

1839–1849.

34. Vale RD, Reese TS, Sheetz MP. Identification of a novel force-

generating protein, kinesin, involved in microtubule-based motility. Cell

1985;42:39–50.

35. Endow SA, Higuchi H. A mutant of the motor protein kinesin that

moves in both directions on microtubules. Nature 2000;406:913–916.

36. Gill SR, Schroer TA, Szilak I, Steuer ER, Sheetz MP, Cleveland DW.

Dynactin, a conserved, ubiquitously expressed component of an

activator of vesicle motility mediated by cytoplasmic dynein. J Cell

Biol 1991;115:1639–1650.

37. Murray JW, Wolkoff AW. Assay of Rab4-dependent trafficking on

microtubules. Methods Enzymol 2005;403:92–107.

38. Ichikawa T, Yamada M, Homma D, Cherry RJ, Morrison IE, Kawato S.

Digital fluorescence imaging of trafficking of endosomes containing

low-density lipoprotein in brain astroglial cells. Biochem Biophys Res

Commun 2000;269:25–30.

846 Traffic 2008; 9: 833–847

Murray et al.

39. Petiot A, Faure J, Stenmark H, Gruenberg J. PI3P signaling regulates

receptor sorting but not transport in the endosomal pathway. J Cell Biol

2003;162:971–979.

40. Murphy RF. Analysis and isolation of endocytic vesicles by flow

cytometry and sorting: demonstration of three kinetically distinct

compartments involved in fluid-phase endocytosis. Proc Natl Acad

Sci U S A 1985;82:8523–8526.

41. van Dam EM, Ten Broeke T, Jansen K, Spijkers P, Stoorvogel W.

Endocytosed transferrin receptors recycle via distinct dynamin and

phosphatidylinositol 3-kinase-dependent pathways. J Biol Chem 2002;

277:48876–48883.

42. Malide D, Cushman SW. Morphological effects of wortmannin on the

endosomal system and GLUT4-containing compartments in rat adipose

cells. J Cell Sci 1997;110:2795–2806.

43. Kurashima K, Szabo EZ, Lukacs G, Orlowski J, Grinstein S. Endosomal

recycling of the Naþ/Hþ exchanger NHE3 isoform is regulated by the

phosphatidylinositol 3-kinase pathway. J Biol Chem 1998;273:20828–

20836.

44. Klopfenstein DR, Tomishige M, Stuurman N, Vale RD. Role of

phosphatidylinositol(4,5)bisphosphate organization in membrane trans-

port by the Unc104 kinesin motor. Cell 2002;109:347–358.

45. Dranoff JA, McClure M, Burgstahler AD, Denson LA, Crawford AR,

Crawford JM, Karpen SJ, Nathanson MH. Short-term regulation of bile

acid uptake by microfilament-dependent translocation of rat ntcp to the

plasma membrane. Hepatology 1999;30:223–229.

46. Krutzik PO, Clutter MR, Nolan GP. Coordinate analysis of murine

immune cell surface markers and intracellular phosphoproteins by flow

cytometry. J Immunol 2005;175:2357–2365.

47. Morell AG, Irvine RA, Sternlieb I, Scheinberg IH, Ashwell G.

Physical and chemical studies on ceruloplasmin. V. Metabolic stu-

dies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968;243:

155–159.

48. Weigel PH, Yik JH. Glycans as endocytosis signals: the cases of the

asialoglycoprotein and hyaluronan/chondroitin sulfate receptors. Bio-

chim Biophys Acta 2002;1572:341–363.

49. Oka JA, Weigel PH. Recycling of the asialoglycoprotein receptor in

isolated rat hepatocytes. Dissociation of internalized ligand from

receptor occurs in two kinetically and thermally distinguishable com-

partments. J Biol Chem 1983;258:10253–10262.

50. Stockert RJ, Potvin B, Tao L, Stanley P, Wolkoff AW. Human hepatoma

cell mutant defective in cell surface protein trafficking. J Biol Chem

1995;270:16107–16113.

51. WatermanH, YardenY.Molecularmechanisms underlying endocytosis and

sorting of ErbB receptor tyrosine kinases. FEBS Lett 2001;490:142–152.

52. Luini A, Ragnini-Wilson A, Polishchuck RS, De Matteis MA. Large

pleiomorphic traffic intermediates in the secretory pathway. Curr Opin

Cell Biol 2005;17:353–361.

53. Mukherjee S, Ghosh RN, Maxfield FR. Endocytosis. Physiol Rev 1997;

77:759–803.

54. Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs, and SNAREs

work together to mediate the intracellular destination of a transport

vesicle. Dev Cell 2007;12:671–682.

55. Bananis E, Murray JW, Stockert RJ, Satir P, Wolkoff AW. Regulation of

early endocytic vesicle motility and fission in a reconstituted system.

J Cell Sci 2003;116:2749–2761.

56. Dillman JF III, Pfister KK. Differential phosphorylation in vivo of

cytoplasmic dynein associated with anterogradely moving organelles.

J Cell Biol 1994;127:1671–1681.

57. Okada Y, Sato-Yoshitake R, Hirokawa N. The activation of protein

kinase A pathway selectively inhibits anterograde axonal transport of

vesicles but not mitochondria transport or retrograde transport in vivo.

J Neurosci 1995;15:3053–3064.

58. Stockert RJ, Haimes HB, Morell AG, Novikoff PM, Novikoff AB,

Quintana N, Sternlieb I. Endocytosis of asialoglycoprotein-enzyme

conjugates by hepatocytes. Lab Invest 1980;43:556–563.

59. Hudgin RL, Pricer WE Jr, Ashwell G, Stockert RJ, Morell AG. The

isolation and properties of a rabbit liver binding protein specific for

asialoglycoproteins. J Biol Chem 1974;249:5536–5543.

60. Murray JW, Bananis E, Wolkoff AW. Immunofluorescence micro-

chamber technique for characterizing isolated organelles. Anal Bio-

chem 2002;305:55–67.

61. Murray JW, Wolkoff AW. In vitro motility system to study the role of

motor proteins in receptor-ligand sorting. Methods Mol Biol 2007;392:

143–158.

Traffic 2008; 9: 833–847 847

Endocytic Segregation In Vitro