The Cell's Muscles and Bones

By Torsten Wittmann, UCSF

Cell movement begins with lamellipodia. A thin sheet of actin filaments (light purple) that
stretches out to the cell's periphery, lamellipodia generate pushing forces that drive the cell
forward. Microtubules (cyan) can barely penetrate this actin network, but they direct cell motility
in other ways, such as controlling cell adhesion and acting as the cell's internal compass.
Image: A human HaCat keratinocyte responds to epidermal growth factor by rapidly forming a
lamellipod around most of its perimeter. The cell was fixed and processed within minutes after
EGF addition. F-actin is stained with fluorescently labeled phalloidin (light purple), and
microtubules are labeled with an antibody (cyan). DNA dye stains the nucleus dark purple.
 Mind the Gap (By Torsten Wittmann, UCSF)
Keratinocytes are the major cell type in the outer layer of skin. When skin is punctured,
keratinocytes move together in sheets to close an open wound. Here a tidal surge of actin
floods forward as the keratinocytes fill in a "wound" in a culture dish. Filopodia or
"microspikes" extend from the lamellipodia and membrane ruffles at the cells' leading edge,
probing the open space before collapsing as the lamella periodically retract.
Movie: HaCat keratinocytes were grown to a confluent cell monolayer on a glass coverslip,
and a free edge was generated by removing half of the monolayer with a razor blade. The
cells respond by polarization and persistent migration into the "wound" without losing cell-
cell contacts. The time-lapse sequence spans ~3 hours and was acquired by differential
interference contrast microscopy.
 Fire the Filopodia
By Tom Deerinck, NCMIR
In contrast to the sheet-like lamellipodia, filopodia are thin finger projections packed with
tight parallel bundles of filamentous or "F"-actin (red). Filopodia are generally thought to
act as cellular "antennae" probing the cell's microenvironment, but filopodia are also
involved in constructing cell-cell adhesions and guiding growing dendrites to
chemoattractants
 Actin Nanomotors
By Marlene Vinzenz, Jan Mueller, and Vic Small, IMBA, Austria
Cell motility is driven by coordinated actin polymerization at the cell's leading edge.
However, it is still fiercely debated exactly how actin generates force to move a cell. The
fine structure of filaments revealed by electron microscopy is exquisitely sensitive to the
preparative methods used, and thus, various models have been proposed. Bringing
together information from electron microscopy, live-cell imaging techniques, and super-
resolution microscopy will be necessary to construct a definitive model.
Image: A cryo-EM tomogram image of a filopodium in a vitreously frozen, live goldfish
fibroblast, showing the actin bundle and the enveloping membrane.
 Bessel Beam Bedazzles
By Liang Gao, Betzig Lab, Janelia Farm
To generate a filopodium on the cell surface, parallel actin filaments nucleate at a small
patch of membrane and then thrust forward like a dart gun. On making contact with
another cell or extracellular matrix proteins, the filopodium may mature into a focal
adhesion, forming a more stable anchor and providing traction necessary for cell
motility.
 I Heart Actin
By Francesco S. Pasqualini, Josue A Goss,
Yvonne S. Aratyn, and Kevin Kit Parker, Harvard
University

Adhesion exerts a powerful influence on a cell's behavior, controlling not only motility and
structure but also growth and survival. The cardiomyocytes shown here adopt distinct shapes
dictated by adhesion onto surfaces coated with fibronectin— an extracellular matrix protein that
anchors cells by binding adhesion receptors. Cardiomyocytes are composed of a highly elaborate
polymer network, primarily actin filaments (green), which contract by the precise alignment and
binding of molecular motors. This type of contraction underlies the mechanical function of the
heart, but also the crawling and migration of other cells.
Image: These cardiomyocytes are derived from embryonic stem cells. Actin (green) and
sarcomeric α-actinin (red) are immunolabeled with Alexa 488-phalloidin and GAM-546/anti- α-
actinin, respectively. DAPI stains DNA blue. Cells were imaged with a Zeiss LSM 510 live
scanning confocal microscope
 The Lone Ranger
By Tom Deerinck, NCMIR
Kupffer cells are specialized macrophages that patrol tiny vessels in the liver called
sinusoids, recycling old red blood cells and ingesting pathogens. The endothelium of
these vessels is perforated with large holes, allowing the Kupffer cells to migrate into
liver tissue at sites of inflammation and damage.
 Primitive Streak
By Anna-Katerina Hadjantonakis, Sloan-Kettering Cancer Center
Cell motility drives key events in development. During gastrulation, epiblast cells undergo an
epithelial-to-mesenchymal transition to generate migratory cells that form the mesoderm and
definitive endoderm. This transition takes place at a structure, called the primitive streak, which
forms at the posterior (or tail) end of the embryo. Cells adopt different fates in the embryo
depending on where and when they leave the streak. The first cells to exit the streak give rise to
extraembryonic, cardiac, and cranial mesoderm, whereas later cells give rise to the lateral plate
and paraxial mesoderm. Cells exiting the anterior streak give rise to definitive endoderm, node,
and midline
 Wrapping Up the Embryo
By Stephanie Nowotarski, Mark Peifer, University of North Carolina
Later on in development, lateral epidermal epithelial cells migrate towards the dorsal midline,
where they meet up with contralateral partners to enclose the embryo in skin. This video follows
this process in a Drosophila embryo. The UAS-GAL4 system drives fusion proteins of both actin
(red) and the actin regulator Enabled (green) in stripes. Enabled localizes to the tips of filopodial
protrusions produced by the cells at the leading edge (yellow arrows); overexpression of Enabled
promotes excessive filopodia formation with a fan-like morphology (yellow arrowheads) and also
induces protrusions from more ventral cells. The video was acquired at 100X with a Visitech VT
Hawk confocal microscope.
 Growth Cone
By Aih Cheun Lee and Daniel
Suter, Purdue University
Perhaps the most spectacular
feat of cell motility is wiring the
complex patterns of neuronal
connectivity in the brain. A
developing axon navigates
towards its synaptic target with
the help of the "growth cone"
at its tip. The dynamics of the
growth cones' actin (red) and
microtubule (green)
cytoskeleton steers the axon
and is essential for directional
migration.

Image: Here a neuronal growth cone from an Aplysia bag cell is labeled for F-actin (red) and
microtubules (green). The image was taken with a 60x 1.4 NA oil immersion objective, plus
additional 1.5x magnification on a Nikon TE2000 inverted microscope (Roper Scientific
Cascade II CCD camera; 2200x total magnification). Metamorph 7.0 was used for microscope
control and image acquisition. The image was processed with Adobe Photoshop CS. Image is
from Suter and Miller (2011), Progress in Neurobiology, 94:91–101.
 A Dangerous Transition (By Anne Weston, Cancer Research UK)
Just as embryonic cells undergo an epithelial to mesenchymal transition during
gastrulation, cancer cells can reactivate this developmental program to become motile
and metastasize to other tissues. During this "EMT," adherens junction components,
such as E-cadherin and β-catenin, are targeted for destruction, while the activation of the
GTPases Cdc42 and Rac1 favors the formation of lamellipodia and filopodia, migration,
and invasion.
Image: Cells from culture were imaged with a JEOL 6700 Field Emission Scanning Electron
Microscope, and then false colored with Adobe Photoshop (scale bar: 10 μm).
 Blebbing Goes Legit
By Anne Weston, Cancer Research UK
Lamellipodia are not the only way for cells to get about— plasma membrane blebs can also drive
cell migration. Once thought to be restricted to dying cells, these spherical membrane
protrusions are now thought to be particularly important in tumor cells, which have been shown
to switch between the lamellipodia- and bleb-based motility.
Image: Cells from culture were imaged with a JEOL 6700 Field Emission Scanning Electron Microscope, and
then false colored with Adobe Photoshop
 Walking Through Walls
By Elliott Hagedorn, David Sherwood, Duke University
Whether a tumor cell migrates into the bloodstream or leukocyte crawls out to
attack pathogens, migrating cells often confront barriers. Invertebrate models
offer a chance to characterize the molecular details of barrier crossing. For
example, during uterine-vulval development in C. elegans, the basement
membrane (blue) that separates these two tissues slides open to create a
passageway for mating and egg-laying.
The Beat Goes On
Scanning electron microscopy image of a single multiciliated cell
(MCC) from the Xenopus epidermis.
Actin Up
By Chanjae Lee and John Wallingford, Howard Hughes Medical Institute & Section of Molecular Cell and
Developmental Biology, University of Texas
The central nervous system (CNS) in most vertebrates forms initially as a flat sheet of cells, which subsequentl
rolls up and fuses shut to form the hollow neural tube, which is the precursor to the CNS. The enriched apical
actin in the closing neural tube (shown in green in the image) is central to cell shape changes that contribute to
the rolling up process.
Image: Color micrograph showing a cross-sectional (transverse) view of the closing neural tube in a Xenopus
embryo. Actin is shown in green.
Looking for Closure
By John Wallingford, Howard Hughes Medical Institute & Section of Molecular
Cell and Developmental Biology, University of Texas
A more dynamic view of neural tube closure, as described in the previous slide.
Image: Movie showing a dorsal view of neural tube closure in a salamander
embryo
http://www.cell.com/cell_picture_show-tubes2?
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Zippering Up the Embryo
By R'ada Massarwa and Lee Niswander, University of Colorado, Denver
Advances in imaging tools and techniques have allowed scientists to image in real-time
embryonic development in many model organisms, pushing the boundaries of what can be
visualized. These advances have also contributed to overcoming some of the particular
challenges inherent to following development in organisms that undergo gestation in utero.
As seen here in a mouse embryo, the dynamics of the second step of neural tube closure–
whereby the two edges of the cell sheet or neural fold "zip" together to form a closed tube–
can be visualized in great detail. See also Massarwa and Niswander, 2013.
Image: Movie showing neural tube closure in a mouse embryo by time-lapsed confocal
imaging. Actin is shown in green; time is in hours. Scale bar, 20 μm.
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Waste Management
By Henk van Veen and Tim J. Schuijt, University of Amsterdam
In vertebrates, the mesenephros (or middle kidney) forms in the developing embryo as an
excretory organ. Structured and functioning similarly to the network of tubules called nephrons in
the adult kidney, the mesenephros filter out waste and maintain proper pH, pressure, and balance
of water to salt and metabolites in the blood. Despite its similarity to the kidney, the mesenephros
only becomes part of the adult kidney in fish and amphibians. It is entirely replaced by a
permanent kidney (metanephros) in reptiles, birds, and mammals. Interestingly, although
mesenephros is lost entirely in female animals, some of the tubules in male animals go on to
become parts of the male reproductive system.
Image: Whole-mount immunostaining with E-Cadherin (green) antibody imaged under a
stereoscope of tubules in the developing mesonephros of a day 6.5 chick embryo
The Milk Route
By Andrew J. Ewald, Johns Hopkins University
The mammary gland, which produces milk, is composed of a dense network of ducts that
gradually forms after birth through a complex process of budding, invasion, and branching. At
birth, the system is a rudimentary duct tree, but in response to hormones, cells in the ducts
proliferate and migrate, allowing the ducts to elongate and invade through the mesenchyme
layer of mammary tissue into the mammary fat pad, where they then begin to branch. Puberty
brings further structural changes to the system of ducts, whereas pregnancy induces the
formation of alveolar structures within the ducts for milk production.
Image: 3D reconstruction of a z stack collected using a spinning disk confocal microscope
showing EGF-induced branching morphogenesis of primary mammary epithelium in 3D Matrigel
culture. Epithelial cells in green; actin in red; nuclei in blue.
A Collective Effort
By Robert J. Huebner and Andrew J. Ewald, Johns Hopkins University
Cell proliferation and alterations in the shape of the tissue that they form drive the elongation
and branching of the tubules that make up the mammary duct system. These tissue
alterations rely on dramatic collective and coordinated movements of the cells within the
ducts.
Image:Time-lapse spinning disk confocal microscopy showing the collective migration of
luminal (red) and myo-epithelial cells (green) during mammary epithelial branching
morphogenesis in 3D Matrigel culture
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