Macropinocytosis-mediated membrane recycling drives neural crest migration by delivering F-actin to the lamellipodium

Macropinocytosis-mediated membrane recycling drives

neural crest migration by delivering F-actin to

the lamellipodium

Yuwei Li

, Walter G. Gonzalez

, Andrey Andreev

, Weiyi Tang

, Shashank Gandhi

, Alexandre Cunha

b,c



David Prober



, Carlos Lois

, and Marianne E. Bronner

a,1



Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;

Center for Advanced Methods in Biological Image

Analysis, Beckman Institute, California Institute of Technology, Pasadena, CA 91125; and

Center for Data-Driven Discovery, California Institute of

Technology, Pasadena, CA 91125

Contributed by Marianne E. Bronner, September 22, 2020 (sent for review June 8, 2020; reviewed by Angela Nieto and Tatjana Piotrowski)

Individual cell migration requires front-to-back polarity mani-

fested by lamellipodial extension. At present, it remains debated

whether and how membrane motility mediates this cell morpho-

logical change. To gain insights into these processes, we perform live

imaging and molecular perturbation of migrating chick neural crest

cells in vivo. Our results reveal an endocytic loop formed by circular

membrane flow and anterograde mov

ement of lipid vesicles, result-

ing in cell polarization and locomotion. Rather than clathrin-mediated

endocytosis, macropinosomes enc

apsulate F-actin in the cell body,

forming vesicles that translocate via microtubules to deliver actin to

the anterior. In addition to previously proposed local conversion of

actin monomers to polymers, we demonstrate a surprising role for

shuttling of F-actin across cells for lamellipodial expansion. Thus, the

membrane and cytoskeleton act in concert in distinct subcellular com-

partments to drive forward cell migration.

membrane recycling

|

actin turnover

|

macropinocytosis

|

cell migration

|

neural crests

ell migration is central to embryogenesis, organogenesis, and

cancer metastasis (1, 2). Individual migrating cells rapidly

change their shape via cycles of protrusion and retraction (3, 4),

raising the question of how a cell distributes its limited amount

of membrane to maintain polarized morphology without affect-

ing membrane integrity. Early studies, based on observing the

movement of cross-linked antigen on the cell surface, proposed a

“

membrane flow

”

model to explain the role of membrane recy-

cling during cell locomotion (5

–

8). This two-dimensional (2D)

model posits that cells undergo clathrin-mediated endocytosis in

the rear of the cell, which generates anterograde flow of lipid

vesicles and retrograde flow of membrane (Fig. 1

). Such a

retrograde membrane flow could enable membrane proteins to

generate traction forces against the extracellular matrix (ECM)

to push the cell forward; thus, this model in theory explains how

mechanical forces could drive cell migration (9, 10). However,

other research presents conflicting data (11), likely due to the

use of different experimental systems and cell tagging reagents.

Moreover, most studies of individual cell motility use cultured

cells or simple organisms, such that little is known about how

membrane and vesicle motion coordinate to influence three-

dimensional (3D) morphological changes of migrating cells in

higher vertebrates.

In addition to the lipid portion of the plasma membrane that

maintains fluidity of the cell boundary, the underlying cytoskel-

eton provides rigidity to the cell surface (12). A

“

treadmilling

”

model was used to explain cytoskeletal regulation of lamellipo-

dial function (13, 14). According to this model, actin polymeri-

zation at the cell

’

s leading edge and actin depolymerization at

the back of the network (Fig. 1

) cause relative displacement to

the cytosol, which subsequently

“

pulls

”

the rounded cell body.

Yet, it is unclear whether and how actin turnover on the cell

’

basal side is coupled with other actin pools and membrane flow

throughout the cells.

To address these long-standing cell biology questions in vivo,

we directly visualize membrane and cytoskeletal behaviors in

migrating neural crest cells at the trunk level of chicken embryos.

The neural crest is one of the most migratory of embryonic cell

types (15), initiating movement via an epithelial to mesenchymal

transition from the neural tube (16). These multipotent cells

then migrate throughout the periphery as individuals (17, 18),

differentiating into diverse cells types including peripheral neu-

rons, glia, and melanocytes of the skin (19). As neural crest-

derived cells are prone to give rise to adult cancers, including

melanoma, neuroblastoma, and gliomas, their innate migratory

mode appears to be recapitulated during cancer metastasis (16).

By combining live imaging with quantitative analysis, we extract

dynamic molecular and cellular information about cell migration

and utilize perturbation approaches to challenge it.

Results

As neural crest cells are a highly migratory cell type that navi-

gates through a complex environment, they represent an ideal

Significance

Membrane and cytoskeletal dynamics are critical to cell motil-

ity. Extensively studied in cell culture, their roles in cell move-

ment in vivo are less understood, especially in higher vertebrates.

We use dynamic imaging to visualize membrane and cyto-

skeletal behavior in migrating neural crest cells in living tissue.

We found that forward movement of individual neural crest

cells is accompanied by circular membrane flow, from anterior-

to-posterior apically and posterior-to-anterior basally, coupled

with internalization of lipid vesicles via macropinocytosis in the

soma. Macropinosomes become wrapped with actin, then un-

dergo anterograde translocation via microtubules toward the

lamellipodium, resulting in its expansion. We elucidate how

actin dynamics and membrane flow are interacted to drive

forward locomotion of individual cells.

Author contributions: Y.L. and W.G.G. designed research; Y.L., W.T., and S.G. performed

research; Y.L., W.G.G., and A.A. contributed new reagents/analytic tools; Y.L., W.G.G.,

A.A., A.C., D.P., and C.L. analyzed data; and Y.L., W.G.G., and M.E.B. wrote the paper.

Reviewers: A.N., Instituto de Neurociencias de Alicante, Consejo Superior de Investiga-

ciones Científicas

–

Universidad Miguel Hernández; and T.P., Stowers Institute for

Medical Research.

Competing interest statement: M.E.B. and A.N. are listed as coauthors on a 2020 Consen-

sus Statement. They did not collaborate directly on the paper.

This open access article is distributed under

Creative Commons Attribution-NonCommercial-

NoDerivatives License 4.0 (CC BY-NC-ND)

To whom correspondence may be addressed. Email: mbronner@caltech.edu.

This article contains supporting information online at

https://www.pnas.org/lookup/suppl/

doi:10.1073/pnas.2007229117/-/DCSupplemental

First published October 21, 2020.

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Fig. 1.

Circular membrane flow across migrating cells. (

) The 2D

“

membrane flow

”

model from top view. Endocytosis at the posterior end produces vesicles

that move toward the anterior end (arrows inside the cytoplasm); these vesicles integrate into the lamellipodium and then translate into retrograde

membrane flow (arrows outside the cell). (

) The 3D

“

treadmilling

”

model from lateral view. F-actin (red) is distributed underneath the plasma membrane. In

the basal side of the lamellipodium, actin displays a net polymerization toward the cell

’

s anterior end.

“

−

”

and

“

”

represents depolymerization and po-

lymerization end of actin, respectively. (

) Schematic illustration of explant culture and imaging. An

∼

500-

m-thick transverse slice is dissected through the

trunk of virally labeled chicken embryos for confocal time-lapse imaging. Successive movies of individual migrating cells are collected at differe

nt positions

from the apical to the basal sides of the cell. For quantitative analysis, a coordinate system is used in which 0 denotes the center of the cell body. (

) Live

imaging reveals a stereotypical fashion of cell migration (

Top

). A cell protrudes the lamellipodium (red arrow) and then progressively retracts its body (yellow

arrow) toward the anterior end. The cell surface is computationally segmented with each optical slice pseudocolored. The surface area of individual seg-

mented slices is measured, and the results are presented in

SI Appendix

, Fig. S1

and

Middle

) Top view. (

Bottom

) Lateral view. (Scale bar: 5

m.) (

–

)

Quantification of membrane flow based on the photo-conversion experiment (see

SI Appendix

, Fig. S1

–

for details). On the basal side, anterior intensity of

the red fluorescence is higher following photo-conversion (t

15 s) (rank sum test in frame 6,

0.001,

7 cells) (

), suggesting anterior membrane flow

(Schematic,

). This scenario is reversed on the apical side (rank sum test in frame 6,

0.001,

8 cells) (

and

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system to study the movement of single cells in an in vivo context.

We take advantages of individual cell labeling and dynamic im-

aging of chicken embryos to visualize intracellular events during

active cell migration.

Circular Membrane Flow Across Migrating Cells.

Using retrovirally

mediated cell labeling in living slices of avian embryos (Fig. 1

)

(18, 20, 21), we imaged Farnesylated-YFP

–

expressing cells to

capture membrane dynamics, with high spatial (lateral: 0.076

per pixel; axial: 1

m per pixel) and temporal (22 s per frame)

resolution (Fig. 1

SI Appendix

,Fig.S1

,andMovie S1

). We

found that individual trunk neural crest cells in vivo migrate

similarly to many other cell types including fibroblasts in vitro;

first, there is extension of the lamellipodium, followed by retrac-

tion of the cell body. Consistent with this, similar cell morphology

was observed in frozen tissue sections (

SI Appendix

, Fig. S1

). As

a simple way to test for membrane recycling, we examined changes

in cell volume. Although we observed remarkable changes in the

surface area at different apicobasal layers, these changes occurred

in opposite directions and thus canceled out (

SI Appendix

, Fig.

and

). As a result, the average individual surface area and

the total volume (sum of the surface area) of the neural crest cells

remained relatively constant over the time of visualization (

Appendix

,Fig.S1

and

), in line with the possibility of

membrane recycling.

To directly assess membrane motion, we conducted a photo-

conversion assay on cells expressing Farnesylated-Dendra2, a

protein that irreversibly turns from green to red fluorescence

after 405-nm laser excitation (22). This approach highlights a

small portion of the cell membrane for estimating the velocity of

membrane flow. Illumination of a small region on the basal

surface of the cell revealed membrane flow toward the anterior

end (Fig. 1

and

SI Appendix

, Fig. S1

and

, and

Movie S2

Conversely, the same experiment focusing on the apical side of

the cells revealed posterior membrane movement (Fig. 1

and

SI Appendix

, Fig. S1

, and

Movie S2

). These results contrast

with the conventional model assuming retrograde membrane

motion on both sides (7). Instead, it suggests circular membrane

motion in a fashion that the membrane moves toward the an-

terior on the basal side and toward the posterior of the cell on

the apical side.

Anterograde Flow of Type I Vesicles Expands the Lamellipodium.

While the above scenario explains the maintenance of mem-

brane integrity, it raises the question of how migrating cells attain

polarity with more membrane material in the anterior for lamel-

lipodial expansion (7). Answering this question requires deter-

mining the mobility of lipid vesicles in the cytoplasm (Fig. 2

and

Movie S3

). We noted that vesicle size appeared to correlate with

their position and speed (

SI Appendix

, Fig. S1

–

): Large vesi-

cles (diameter more than 1.0

m) were located on the apical side

and exhibited slow movement; however, fast-moving small vesicles

(diameter between 0.5 and 1.0

m) were in distinct layers along

the apical-basal axis. The relatively static state of large vesicles was

inconsistent with a direct role in controlling lipid flow. Therefore,

we focused on small vesicles and partitioned them into two classes

depending on their site of origin: type I derived from the apical

side of the cell body and type II derived from the lamellipodium

(Fig. 2

and

We first explored the kinetic behaviors of type I vesicles based

on a coordinate system defined according to the cell

’

s geometry

(Fig. 1

). In the imaging plane, the anterior-posterior (major)

axis of the cell was defined as the

axis with the

axis perpen-

dicular to it. The

axis runs along the apical-basal axis (or-

thogonal to the image plane) of the cell. Decomposition of

vesicle trajectories revealed that the vesicles fell into two clusters

with distinct kinetics in the anterior-posterior direction (Fig. 2

and

): 45% of the vesicles migrated long distance toward the

basal/anterior end, whereas 55% moved within the cells body

(Fig. 2

and

). Along the

axis, vesicles were generally dis-

placed in an apical to basal direction (Fig. 2

What are the consequences of the distinct migration trajec-

tories of these two subgroups of type I vesicles on cell shape

changes? We addressed this question using a custom software

tool to quantify the relationship between the final destinations of

vesicles and the protrusion of local cell membrane. Here, we

followed the trajectories of individual vesicles; upon their fusion

with the cell membrane, we calculated the change in the size of

the local membrane (based on the intensity of Membrane-YFP)

as a function of distance from the site of vesicle fusion. In this

way, we found that vesicles moving into the front end fused with

the membrane and expanded the lamellipodium (Fig. 2

–

);

however, those vesicles restricted to the cell body did not affect

local cell morphology upon integrating into the membrane

(Fig. 2

–

As vesicle trajectories correlate with their contribution to cell

morphogenesis, we next asked how vesicle motility is controlled

(23). Conceptually, vesicle motion might reflect their bound versus

unbound state to the cytoskeleton (24). Alternatively, these vesi-

cles may always be bound to the cytoskeleton that, in turn, impacts

vesicle dynamics. Visualizing the interaction between vesicles and

cytoskeletal components in space and time offers a direct means to

test between these two possibilities. To that end, we imaged dis-

tinct layers in the cells expressing Utrophin-scarlet, which binds to

F-actin. The results showed a stereotypical distribution of F-actin

subgroups with actin bundles on the basal region versus actin

patches and cortical actin on the more apical side (

SI Appendix

Fig. S2

) (25). While actin bundles are commonly used as tracks

for endocytic transportation (26), their basal localization in neural

crest cells cannot account for the apical-basal movement of type

I vesicles.

Microtubules Mediate Anterograde Flow of Type I Vesicles.

subsequently assessed the spatial organization of microtubule

(MT) using 2G4-GFP, an intrabody that specifically and non-

invasively targets tubulin (27, 28). This labeling revealed a cable-

like MT network, extending from the microtubule organizing

center at the cell

’

s apical/posterior region to the basal/anterior end

(

SI Appendix

,Fig.S2

). Time-lapse imaging and colocalization

analysis between MTs and vesicles further suggested that lipid

migration occurred along this network (Fig. 3

and

and Movie

S4). Notably, we also observed vesicle motion on many MTs that

filled the entire cell body (

Movie S5

). However, these vesicles

moved more slowly than those associated with the lamellipodium

(Fig. 3

). Such a patterned relationship between vesicle mobility

and MT morphology supports the possibility that vesicles are in a

bound state and their trajectories are substantially influenced by

MT morphology.

Since the morphology of MTs is controlled by their growth ori-

entation in many tissue systems (29), we tracked the MT plus end

binding protein EB3 in neural crest cells to determine whether

there is a correlation between MT dynamics and vesicle mobility

(Fig. 3

and Movie S6

). Following an established protocol (30), we

measured the orientation of EB3 signal with respect to position in

the cell (Fig. 3

–

). As predicted, MTs showed less directional

flow in the cell body than in the lamellipodium (Fig. 3

–

). This

helps to explain the differential mobility of the two subpopulations

of type I vesicles. Based on these findings, we propose that MTs

mediate anterograde flow of type I v

esicles; this, in turn, integrates

with circular membrane flow to create an endocytic cycle.

Type I Vesicles Are Noncanonical Macropinosomes Transporting F-Actin

into the Lamellipodium.

Given the stereotypical mobility of type I

vesicles and their contributions to the cell

’

s morphological

changes, we next explored their nature. Clathrin-mediated endo-

cytotic vesicles are typically of diameters smaller than 0.2

m(31),

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Fig. 2.

Anterograde flow of type I vesicles expands the lamellipodium. (

) A segmented view of a migrating cell with its trajectory color-coded according to

time (thick line). All of the vesicles inside the cell are segmented (yellow dots), and their trajectories are mapped (thin lines). (

Inset

) Vesicle motion is nor-

malized relative to the movement of the center of the cell body. (Scale bar: 5

m.) (

and

) Two types of vesicles from top (

) and lateral (

) view. The lengths

of the net displacement vectors of type I and II vesicles are proportional to their moving distance inside the cell. The schematics illustrate vesicle trajectories.

(Scale bars: 5

m.) (

and

) Two subpopulations of type I vesicles from top (

) and lateral (

) view. Their net displacement vectors are presented with the ones

moving to the front and the other moving inside the cell body. (Scale bars: 5

m.) (

) Trajectory analysis of type I vesicles based on the coordinate system in

Fig. 1

. The trajectories are color-coded according to their destinations along the anterior-posterior axis (

axis) (

15 and 18 for green and red, respectively).

Green tracks show upward shift in this direction, meaning that these vesicles move toward the cell

’

s front end. Along the

axis, upward shift of many green

lines reflects lamellipodial extension toward the cell

’

s left side.

3 cells. (

) Distance analysis confirms that the vesicles moving to the cell front show

maximal displacement (along the

axis).

15 and 18 for vesicles moving to the front and in the cell body, respectively.

3 cells (rank sum test,

0.724;

0.001;

0.018). (

–

) Vesicles moving to the cell front expand the lamellipodium. (

) Two representative time frames showing vesicle (arrow) fusion

into the membrane causes membrane protrusion. (

–

) Quantification (

Methods

). (

) The average intensity near the vesicle before fusion. The red contour

shows the approximate cell membrane. The dashed and solid black contours show fluorescent signal inside and outside of the cells, respectively. (

) Average

intensity change due to vesicle fusion. (

–

) The average maximum intensity changes as a function of distance from the site of vesicle fusion. Signal intensity

decreases

in the intracellular regions while increases in the extracellular regions (

); this is not observed in the random dataset (

). (

) Summary of the results

and

(a: intracellular changes, b: extracellular changes, c: extracellular changes in the random dataset) (rank sum test, **

0.001,

31 pixels

representing the average across 36 vesicles). (

–

) Vesicle motion in the cell body does not impact cell morphology. (

) Two representative time frames. The

same analysis in

–

is applied here. Note that the extracellular changes of the membrane (b) in

is not significantly different to intracellular changes (a) and

the random dataset (c).

3 cells (rank sum test,

31 pixels representing the average across 36 vesicles). n.s., not significant. (Scale bars for

m.)

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whereas type I vesicles are much larger, having diameters of 0.5

–

SI Appendix

, Fig. S1

). Thus, these vesicles are unlikely to be

clathrin-coated vesicles. Instead, they could either be the products

of phagocytosis (e.g., by ingesting dead cells) or macropinocytosis

(by absorbing fluids) (32). In a long term (up to 15 h) imaging

session with a field of view covering more than 20 cells, only 1 cell

was observed to engulf debris from dead cells (Fig. 4

and Movie

S7); this is insufficient to account for the prevalence of type I

vesicles in all observed cells. Meanwhile, several findings sup-

ported a possible role for macropinocytosis. This endocytic event

is receptor independent but exhibits features of membrane ruf-

fling, such as planar folding or circular extension which then en-

gulfs liquid (33). Indeed, on the apical side of the cell body, we

observed protrusion of the cell membrane into a cup-shaped

structure that closed upon itself; in the subsequent retraction

phase, the closed cup broke down into small vesicles (Fig. 4

and

Movie S8

). To further determine if these vesicles are in bona fide

macropinosomes, we incubated the embryo slices with dextran, a

commonly used vital dye to characterize macropinosome-

mediated endocytosis (34), and observed immediate labeling of

the vesicles (

SI Appendix

,Fig.S3

and Movie S9

An independent assay for macropinocytosis is provided by

inspecting the expression pattern of actin. During macro-

pinocytosis, F-actin forms a belt-shaped band that surrounds the

vesicles to generate contractile forces for vesicle fission, and this

actin ring disappears when vesicles move into the cell (33).

Analogously, we observed that cortical actin in neural crest cells

initially binds to the plasma membrane in the cell body, ruptures

as membrane protrusion retracts, and then associates with the

cup-shaped membrane structure (Fig. 4

and

Movie S10

Fig. 3.

MTs mediate anterograde flow of type I vesicles. (

and

) Live imaging of vesicle movement along MTs (

). (

) Fluorescence colocalization analysis

shows that MT amount (2G4-GFP intensity) is higher in the region enriched with vesicles (Membrane-YFP intensity) compared with in the region without

vesicles (Ctrl) (rank sum test, **

0.001,

3 cells). (

) Speed analysis shows that vesicles in the lamellipodium move faster (rank sum test, **

0.001,

cells). (

) Higher-frequency (collecting a frame every 5 s) imaging of MT plus ends. (Scale bar: 4

m.) (

–

) Approach to measuring the orientation of MT plus

ends (

Methods

). (

) In the region of interest (ROI), individual EB3 signal in

is zoomed in such that its shape can be clearly visualized. (

) For individual EB3

signal, its 2D fast Fourier transform (FFT) representation is calculated. (

) The cross-correlation between the orientation of FFT image and the long axis of cells

is measured. This cross-correlation value represents the relative orientation of EB3 with respect to the cell. (

–

) Distinct flow orientation of MTs in the

lamellipodium and in the cell body. In a randomly selected time frame (

), the approach introduced in

–

is applied to measure the orientation of EB3 flow.

and

, bold black lines are the mean cross-correlation value for all EB3 signal. Gray area signifies SD, which positively correlates with the orientation o

f EB3.

The higher SD in the cell body (

) than in the cell front (

) suggests that EB3 flow in the cell front end is more oriented. (Scale bar in

m.)

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Strikingly, instead of being gradually dissociated from the vesi-

cles, F-actin further aggregated into small patches residing in the

vesicles and moved together with the vesicles to the basal/ante-

rior end (Fig. 4

SI Appendix

, Fig. S3

, and

Movie S10

). Using

phalloidin staining, we confirmed that endogenous F-actin was

wrapped by lipid vesicles (

SI Appendix

, Fig. S3

). Upon arriving

in the lamellipodium, as the lipid components of the vesicles

fused to the membrane, F-actin patches were docked into the

actin module (Fig. 4

–

and Movie S11

We further characterized the relationship between these ves-

icles and actin, and found that F-actin inside the vesicles was

more stable than that outside (

SI Appendix

, Fig. S3

and

Movie S12

). In contrast, Actin-scarlet, labeling both F-actin and

G-actin (actin monomer), was expressed at a lower level inside

Fig. 4.

Type I vesicles are noncanonical macropinosomes transporting F-actin to the lamellipodium. (

) Live imaging in a 612

612

region between

the dorsal neural tube and the notochord fails to detect evidence of phagocytosis. (Scale bar: 20

m.) (

) Live imaging of macropinocytosis. The extending

protrusion in the cell body retracts and translates into vesicles (arrows). (Scale bar: 3

m.) (

) Live imaging on macropinosome wrapping of F-actin and

subsequent transportation. (Scale bar: 4

m). Box: zoom in view of the process of cortical actin breakage into actin patches, association with lipid vesicles and

transportation into the lamellipodium (arrows) (Scale bar: 2

m). (

) Addition of a F-actin patch into the actin bundles in the lamellipodium (green:

Membrane-YFP; red: Utrophin-scarlet). When a vesicle moves to cell front (yellow arrow), its lipid causes membrane protrusion (white arrow); simultaneously,

the actin patch inside this vesicle is released (cyan arrows) and merged with the existing actin bundles. (Scale bar: 1

m.) (

–

) Morphological analysis confirms

lipid expansion of cell membrane (

–

) and F-actin insertion into the lamellipodium (

–

). The same approach in Fig. 2

–

is applied to assess the contribution

of lipid and F-actin to lamellipodial morphogenesis. Focusing on

, increase of Membrane-YFP intensity in the extracellular region (b) suggests that lipid fusion

causes membrane protrusion. Similarly, in

, increase of Utrophin-scarlet intensity in the extracellular region is a sign of F-actin insertion into the lamelli-

podium (for

and

, rank sum test, **

0.001,

31 pixels representing the average of 17 vesicles). (Scale bars in

and

m.)

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the vesicles (

SI Appendix

, Fig. S3

and

and Movie S12

). In a

control experiment, membrane bound Cortactin-scarlet showed

similar expression patterns to total actin (

SI Appendix

, Fig. S3

and

and Movie S12

) (35). Collectively, this strongly suggests

that the vesicles are comprised of low amounts of total actin with

the majority being F-actin and operate as affinity and non-

permeable transporters of F-actin. Hence, cortical actin in the

cell body can transform into actin bundles in the lamellipodium

through vesicular transportation (

SI Appendix

, Fig. S3

). Since

these vesicles are distinct from classic macropinosomes with only

transient actin interactions, we termed them noncanonical

macropinosomes (Fig. 5

Type II Vesicles Are Canonical Macropinosomes Acting Inside the

Lamellipodium.

Next, we examined anteriorly located type II

vesicles (Fig. 2

and

) and demonstrated that they are canonical

macropinosomes. In neural crest cells, some region of the lamelli-

podium folded into the apical side of the cell, further invagi-

nated into the basal side and broke down into vesicles (Fig. 5

and

). This phenomenon resembles planar folding of the

plasma membrane during macrop

ynocytosis (32). Unlike type I

vesicles, these vesicles transi

entlybindtoactinringsduring

their formation (Fig. 5

and Movie S13),acommonfeatureof

classic macropinosomes (33); afterward, they behave similarly

to type I vesicles by moving along the MTs and expanding the

plasma membrane (Fig. 5

–

). These canonical macro-

pinosomes may permit neural crest cells to quickly adjust the

shape and size of the lamellipodium, thus operating as local

control to complement to the global-scale influence of noncanonical

macropinosomes (Fig. 5

Membrane Flow and Vesicle Flow Play Distinct Roles in Shaping the

Lamellipodium.

After determining the orientation of membrane

motion and vesicle motion and their relationship, we segregated

their contributions to cell morphogenesis. Given that MTs

operate as the tracks for vesicle transportation (Fig. 3), they

provide a valuable tool to manipulate vesicle mobility. Impor-

tantly, MT dynamics did not appear to impact membrane flow.

In support of this, we analyzed the movements of membrane,

MTs, and vesicles in the direction of cell migration (Fig. 6

and

SI Appendix

, Fig. S4

and

) and demonstrated these

three processes occurred in a sequential order. This phenome-

non further implies that membrane flow and vesicle flow are

distinct and additive in defining the shape of the lamellipodium.

We functionally tested this possibility by blocking MT poly-

merization with nocodazole (29). Using a photo-conversion assay,

we confirmed that nocodazole did not affect normal circulation of

the plasma membrane (Fig. 6

and

and Movie S14

); hence,

MTs do not directly control membrane mobility. However, several

studies based on dynamic imaging (Fig. 6

) demonstrated a crit-

ical role for MTs in directing vesicle motion. In the treated cells,

vesicles underwent fission (Fig. 6

and Movie S15

)andmoved

more slowly than normal (

SI Appendix

,Fig.S4

). Consistent with

this, vesicles

’

travel distance within the cells was shorter than

normal (Fig. 6

) and these vesicles did not migrate close to the

cell boundary (Fig. 6

and

Movie S16

) to integrate into the

membrane. Although many treated cells extended long processes,

these processes did not grow into a fan-shaped lamellipodium

(Fig. 6

and Movie S1

); instead, they receded into the cells, and

repeated extension/retraction in the adjacent region (Fig. 6

and

and Movie S17

), a phenomenon referred to as cell blebbing (36).

As expected, the aberrant vesicle kinetics and low F-actin com-

position (Fig. 6

) resulted in unsustainable growth of the bulging

region (

SI Appendix

,Fig.S4

In many cell types, directional endocytic trafficking along MTs

is regulated by Rab11, a small GTPase mediating the interac-

tions between vesicles and other components including motor

proteins, coat proteins, and scaffolding proteins (37). In neural

crest cells, Rab11 colocalized with vesicles in neural crest cells

(Fig. 7

–

). Further, cells overexpressing a dominant-negative

mutant form (DN-Rab11) exhibited normal features of MT or-

ganization (Fig. 7

), affording an additional experimental system to

assess the influence of vesicle flow on cell morphogenesis. Within

these cells, vesicles moved at normal speed (

SI Appendix

,Fig.S4

)

but their migration was not as directional as the front-moving ves-

icles in the normal cells (Fig. 7

and

SI Appendix

ig.S4

,and

Movie S18

). This was exemplified by the zigzag shapes of vesicle

trajectories (

SI Appendix

,Fig.S4

) and their shorter moving dis-

tance (Fig. 7

). Compared with the nocodazole-treated cells, the

phenotype of vesicle mobility in t

his scenario was more moderate

and led to relatively small differences in both vesicle destinations

and cell morphology. Due to their normal migration speed, these

vesicles still moved to the cell boundary (Fig. 7

) and promoted

membrane protrusion into the lamellipodium (

SI Appendix

,Fig.

–

). Nevertheless, this event occurred in both cell anterior and

posterior ends (Fig. 7

and

), relating to the more random nature

of vesicle trajectories (

SI Appendix

,Fig.S4

). Under these condi-

tions, the lamellipodial structure was not maintained in one par-

ticular direction but retracted and then formed on the other side of

the cells (Fig. 7

and

SI Appendix

,Fig.S4

), as validated by

mapping angle progression of the lamellipodium (

SI Appendix

,Fig.

–

Finally, since vesicles are generated through macropinocytosis,

we asked whether this process also would be affected by nocodazole

or DN-Rab11 treatment. We did not detect canonical macro-

pinocytosis characterized by membrane folding in either case.

Concomitantly, we noticed cup-

shaped extensions of the non-

canonical macropinocytosis and resultant vesicles in the cell body

(Figs. 6

and 7

). Hence, upon interfering with normal vesicle mo-

bility, canonical macropinocyto

sis is inhibited but noncanonical

macropinocytosis still occurs, albeit

producing vesicles with abnor-

mal kinetics. As a consequence, cells exhibited unsustainable po-

larity (Figs. 6

and 7

) and impaired mobility (

SI Appendix

,Fig.

). Together, these results support the idea that membrane flow,

vesicle fusion, and continuous vesicle flow, respectively, lead to

membrane protrusion, lamellipodial formation, and lamellipodial

maintenance.

Discussion

In this study, we identify a causal relationship between lipid

and cytoskeleton to control cell migration in higher verte-

brates. At the heart of our newly formulated model (Fig. 7

)

lies the interaction between circular membrane motion and

anterograde vesicle flow. Whereas the basal/anterior motion

of the plasma membrane initiates lamellipodial formation, on

the apical side, membrane flows toward the cell body and

internalizes through noncanonical macropinocytosis. These

vesicles transport F-actin along MTs to enhance the actin

module and also shape the lamellipodium. Overlaid on this,

canonical macropinocytosis regionally adjusts lamellipodial

morphology. The whole intracellular recirculating system is

driven by MT growth.

In contrast to the conventional

“

membrane-flow

”

model

bilateral retrograde flow of membrane, we present in vivo and

3D evidence to support anterograde flow of membrane at the

cell

’

s basal side and anterograde flow of vesicles inside the cy-

toplasm. We further show that these two flows sequentially ex-

tend the leading edge at the expense of membrane in the rear,

and this results in a net forward displacement of the cell body.

Such a scenario provides a physical linkage between membrane

mobility and cell polarity, and may explain cell migration in the

absence of traction force. To achieve the balance between cell

shape change and lipid homeostasis, loss of lipid in in the rear is

compensated for by the retrograde membrane flow on the

apical side.

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How is the membrane lipid translated into the cytoplasmic

lipid? Based on the cell morphological analysis, dextran inter-

nalization assay and monitoring of actin behavior, our study

highlights the importance of macropinocytosis throughout the cell

for membrane internalization. We speculate that there are two

advantages of macropinocytosis: (

) independence from specific

receptors and (

) the ability to exert both short-range and long-

range effects on the lamellipodium through division of labor,

providing cells with great flexibility to recycle lipids.

By analyzing actin evolution in neural crest cells in 3D em-

bryos, we demonstrate that F-actin in the rear can directly fuel

this actin pool through active shuttling. Our finding does not

argue against

“

treadmilling

”

model (13, 14); rather it highlights

multiple mechanisms in maintaining actin recirculation. As

these F-actin patches are transported by the noncanonical

macropinosomes, one possibility

is that some actin-associated

proteins incorporate into these vesicles during tail retraction;

upon moving to the leading edge, both components (as an in-

tact complex) thus could immediately participate in construct-

ing the actin machinery. In turn, the organization of F-actin

across the cells influences how ve

sicles sculpt cells at 3D level.

Our data suggest that the periphery of the cell body is wrapped

Fig. 5.

Type II vesicles are canonical macropinosomes acting inside the lamellipodium. (

) Live imaging of the cellular process of type II macropinosytosis. The

cell subjected to live imaging with its migratory trajectory (colored line). The box shows that within the lamellipodium membrane folding and degradation

into vesicles; during this event, the vesicles only transiently bind to F-actin (arrows). The cyan arrows point to the actin rings (t

132 s and t

154 s). (

segmented and lateral view of the same cell presented in

. The folding part of the membrane and the resultant macropinosomes are labeled in red. (

–

)

Lamellipodial extension by type II vesicles, as revealed by the same software tool in Fig. 2

–

. Focusing on

, increase of Membrane-YFP intensity in the

extracellular space (comparing b with a and c) demonstrates that lipid addition to the membrane results in cell protrusion (rank sum test,

0.001,

pixels representing the average of 22 vesicles). (Scale bars in

–

m.) (

) Schematic representation of noncanonical macropinocytosis versus canonical

macropinocytosis.

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Fig. 6.

Blocking MT polymerization interferes vesicle flow and lamellipodial formation. (

and

) Live imaging shows membrane protrusion occurs in ad-

vance of MT flow and vesicle flow (

). A window (yellow) is drawn in the direction of membrane extension, and the evolution of fluorescence signal of

membrane, MT, and vesicles within this window is measured (

B, Upper

shows the sequential movements of the membrane, MT, and vesicles. (Scale bar in

m.) (

and

) Normal membrane flow in the nocodazole-treated cells. The same photo-conversion assay to Fig. 1

is performed. (

) Photo-conversion on

the basal side of the cells expressing Farnesylated-Dendra2 shows the red fluorescence move more quickly to the anterior end than to the posterior end (rank

sum test at frame 4,

0.001,

12 cells). The same analysis on the apical side reveals posterograde membrane flow (rank sum test at frame 4,

0.05,

cells) (

). (

) Live imaging of a treated cell; note that the cell does not form the lamellipodium. In the segmented image, the displacement vectors are more

randomized than the ones moving to the front in the normal cells in Fig. 2

. (Scale bar: 5

m). (

) Live imaging to show vesicle fission (arrows) in the treated

cells. (Scale bars: 2

m.) (

) Trajectory analysis reveals abnormal vesicle movement in the treated cells. In both the medio-lateral (

axis) and anterior-posterior

(

axis) directions, all of the lines extend toward 0 (the center of the cell body), demonstrating that vesicle motion is restricted within the cell body.

(

)

Distance analysis reveals minimal movement of the vesicles along the anterior-posterior axis of the treated cells (

axis), relative to their mobility in the normal

cells in Fig. 2

35 vesicles,

3 cells (rank sum test,

0.001,

0.241;

0.692;

0.004). (

) The treated cells are blebbing. The expanding

region is almost devoid of F-actin (t

132 s, arrow); however, upon shrinkage, F-actin is enriched on the edge (t

176

s, arrow) to exert contraction force.

(Scale bar: 3

m.) (

) Live imaging of noncanonical macropinocytosis in a treated cell. The ruffles on the cell body recede into the cytoplasm accompanied by

vesicle delamination (arrows). The morphologies of these vesicles are not obvious due to fission. (Scale bar: 3

m.)

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Fig. 7.

Blocking Rab11 activity interferes vesicle flow and lamellipodial maintenance. (

–

) Rab11 is colocalized with vesicles. (

) Live imaging to show

Rab11-DsRed labeled puncta (red) is enriched in the vesicles.

illustrates the principle of colocalization analysis. The inner ring is drawn along the border of

Membrane-YFP (green). The red pixels in the inner ring and in the region between the inner and outside rings are measured. The ratio between them is

plotted (

); if the ratio is more than one, it means more Rab11 inside the vesicles than in the cytoplasm. (Scale bar in

m.) (

) MT organization in a DN-

Rab11-2A-mCherry

–

positive cell. Three individual slices of the cell (from basal to apical side) are presented. In the 3D image (

Upper Right

), each individual slice

is pseudocolored. Similar to the untreated cells in

SI Appendix

, Fig. S2

) Live imaging of a DN-Rab11-2A-mCherry

–

expressing cell shows lamellipodial

extension in multiple directions. (

) Net displacement vectors of the cell in

showing that vesicle movement is not directional. (Scale bars in

–

m.) (

)

Trajectory analysis of the vesicles in the DN-Rab11

–

positive cells. Along the

axis, the lines display both upward and downward shifts, the evidence of vesicle

migration to either end of cells.

35 vesicles,

3 cells. (

) Distance analysis shows these vesicles move in shorter distance than the normal ones in Fig. 2

35 vesicles,

3 cells (rank sum test,

0.001,

0.905;

0.543;

0.433). (

) Live imaging of noncanonical macropinocytosis in a DN-

Rab11

–

expressing cell. Similar to both untreated (Fig. 4

) and nocodazole treated cells (Fig. 6

), tail retraction produces small vesicles (arrows). (Scale bar: 3

m.) (

) Schematic summary of the membrane and cytoskeletal processes in controlling cell polarization and locomotion.

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by a layer of cortical actin, which functions like a physical fence

to prevent cell protrusions after l

ipid addition. In contrast, the

lamellipodium is free of cortical actin. This helps to explain

why, despite the existence of vesicles fusion to the membrane in

both the cell body and the lamellipodium, only the latter leads

to morphological change.

Considering that regulation of transcription, translation,

and ubiquitin-mediated prote

in degradation takes signifi-

cant amounts of time that are likely too long to enable rapid

morphological changes in migr

ating cells, recycling and

redistributing of the existing cellular components appears to

be a more direct means to solve this problem. Our study

elucidates the recirculating sy

stem that unifies different cel-

lular components, supporting the idea of

“

efficiency through

simplicity.

”

Identification of a linkage between membrane and

cytoskeletal dynamics in neural crest cells provides a predic-

tive model that can be tested fo

r other motile cells including

cancer cells. Advanced imaging techniques such as multipho-

ton imaging (38), fluorescence lifetime imaging (39), fluores-

cence spectroscopy microscopy (40), second harmonic generation

(41), hyperspectral imaging (42), and light sheet imaging in

the near-infrared II window (43) will provide solutions to this

challenge.

Materials and Methods

Molecular Cloning and Viral Production.

Recombinant RIA plasmids were

cotransfected with Envelop A plasmid into chick DF1 cells in 15-cm culture

dishes using standard transfection protocol (20). When the cells were con-

fluent, the cell culture medium was harvested once per day for 3 d and was

concentrated at 26,000 rpm for 1.5 h. The pellet was dissolved in a minimal

volume of DMEM.

Viral Infection and Explant Culture of Chicken Embryos.

Fertilized chicken eggs

were incubated at 38 °C until embryos reached stages HH11+/12- (44). To

achieve efficient transfection of the neural tube and neural crest cells,

concentrated virus (10

to 10

pfu/mL) were injected into the posterior

neuropore, filling the entire tube. Injected embryos were incubated at

38 °C for 24 h, collected with filter paper carriers, and washed in Ringer

’

solution. Transverse cuts through posterior sclerotomes of the forelimb

region were made every two somites and put into fluorodish-containing

Neurobasal media.

Image Acquisition and Segmentation.

The whole fluorodish was transferred

into the incubation chamber (37 °C and 5% CO

) of a Zeiss LSM 800 inverted

microscope for time-lapse imaging. For all imaging experiments, optical

sectioning was achieved at 1-

m intervals. For long-term imaging of

Faenesylated-YFP

–

expressing cells to detect phagocytosis, 20

/0.8 N.A. ob-

jective was used and digital amplification was set to 0.6. The samples were

excited by 488-nm laser with 0.8% relative power and imaged at 2-min in-

tervals for 12 h. For imaging MT and EB3 dynamics, 63

/1.4 N.A. objective

lens was used and digital amplification was set to 3. The samples were im-

aged at 5-s intervals. 2G4-GFP and EB3-Sarlet were excited by 488-nm laser

with 0.4% relative power and 561-nm laser with 0.7% relative power, rel-

atively. The four-dimensional (4D) images were imported into IMARIS 9.3.

Three-dimensional surface segmentation of cells, 3D spot segmentation, and

4D tracking of vesicles were performed.

Analysis of Lipid Addition to Cell Membrane.

Corresponding to Fig. 2

,the

last frame of every vesicle was identified. The field of view was rotated so

that the membrane nearest the last observed position of the vesicle is

oriented vertically downward (i). Thus, the last observed vesicle position is

at the center and the nearest membrane is at the bottom. The average

across all vesicles is shown in Fig. 2

. The inside of the cell is delineated by

the solid red line marking average membrane intensities above 43 (arbi-

trary units). The resulting membrane extension due to vesicle fusion (J),

calculated by the difference between last frame in which the vesicle was

observed and the next frame. The green contour shows the largest

changes in the membrane, which are mai

nly localized to the extracellular

region (red contour).

Data Availability.

All study data are included in the article and supporting

information.

ACKNOWLEDGMENTS.

We thank Pierre Martineau for sharing reagents

and Beckman Institute Biological Imaging Facility at Caltech for sharing

equipment. We thank the Beckman Institute at Caltech for financial

support to the Center for Advanced Methods in Biological Image Analysis

(A.C.). W.G.G. is supported by the Della Martin Foundation, the American

Heart Association, and the Burroughs Wellcome Fund. S.G. is supported by

the American Heart Association. This project is supported by DE024157 and

R35NS111564 (to M.E.B.).

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