Group I PAKs function downstream of Rac to promote podosome invasion during myoblast fusion in vivo

The p21-activated kinases (PAKs) play essential roles in diverse cellular processes and are required for cell proliferation, apoptosis, polarity establishment, migration, and cell shape changes. Here, we have identified a novel function for the group I PAKs in cell–cell fusion. We show that the two Drosophila group I PAKs, DPak3 and DPak1, have partially redundant functions in myoblast fusion in vivo, with DPak3 playing a major role. DPak3 is enriched at the site of fusion colocalizing with the F-actin focus within a podosome-like structure (PLS), and promotes actin filament assembly during PLS invasion. Although the small GTPase Rac is involved in DPak3 activation and recruitment to the PLS, the kinase activity of DPak3 is required for effective PLS invasion. We propose a model whereby group I PAKs act downstream of Rac to organize the actin filaments within the PLS into a dense focus, which in turn promotes PLS invasion and fusion pore initiation during myoblast fusion.

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Pulsed actomyosin contractions in morphogenesis

F1000Research ◽

10.12688/f1000research.20874.1 ◽

2020 ◽

Vol 9 ◽

pp. 142 ◽

Cited By ~ 1

Author(s):

Ann Sutherland ◽

Alyssa Lesko

Keyword(s):

Cell Shape ◽

Normal Development ◽

Tissue Growth ◽

Model Systems ◽

Cellular Processes ◽

Cytoskeletal Networks ◽

And Migration ◽

Shape Changes

Cell and tissue shape changes are the fundamental elements of morphogenesis that drive normal development of embryos into fully functional organisms. This requires a variety of cellular processes including establishment and maintenance of polarity, tissue growth and apoptosis, and cell differentiation, rearrangement, and migration. It is widely appreciated that the cytoskeletal networks play an important role in regulating many of these processes and, in particular, that pulsed actomyosin contractions are a core cellular mechanism driving cell shape changes and cell rearrangement. In this review, we discuss the role of pulsed actomyosin contractions during developmental morphogenesis, advances in our understanding of the mechanisms regulating actomyosin pulsing, and novel techniques to probe the role of pulsed actomyosin processes in in vivo model systems.

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Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

The Journal of Cell Biology ◽

10.1083/jcb.201508056 ◽

2016 ◽

Vol 212 (2) ◽

pp. 219-229 ◽

Cited By ~ 67

Author(s):

Mo Weng ◽

Eric Wieschaus

Keyword(s):

Cell Shape ◽

Adherens Junctions ◽

Myosin Ii ◽

Cell Junctions ◽

Snail Expression ◽

Quantitative Image ◽

Early Expression ◽

Temporally Correlated ◽

Shape Changes

Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT.

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Direct laser manipulation reveals the mechanics of cell contacts in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418732112 ◽

2015 ◽

Vol 112 (5) ◽

pp. 1416-1421 ◽

Cited By ~ 123

Author(s):

Kapil Bambardekar ◽

Raphaël Clément ◽

Olivier Blanc ◽

Claire Chardès ◽

Pierre-François Lenne

Keyword(s):

Cell Shape ◽

Constitutive Law ◽

Cell Junctions ◽

Scale Model ◽

Tissue Morphogenesis ◽

Cell Contacts ◽

Cell Shape Changes ◽

Shape Changes ◽

Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

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Tendon-derived biomimetic surface topographies induce phenotypic maintenance of tenocytes in vitro

10.1101/2020.07.23.217224 ◽

2020 ◽

Author(s):

Aysegul Dede Eren ◽

Aliaksei Vasilevich ◽

E. Deniz Eren ◽

Phanikrishna Sudarsanam ◽

Urandelger Tuvshindorj ◽

...

Keyword(s):

Gene Expression ◽

Cell Shape ◽

Flat Surface ◽

Flat Surfaces ◽

Tissue Culture Polystyrene ◽

Surface Topographies ◽

And Function ◽

Shape Changes

AbstractThe tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

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Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo

eLife ◽

10.7554/elife.29140 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 28

Author(s):

Yidi Sun ◽

Nicole T Leong ◽

Tommy Jiang ◽

Astou Tangara ◽

Xavier Darzacq ◽

...

Keyword(s):

Quantitative Imaging ◽

Threshold Level ◽

Actin Assembly ◽

Cellular Processes ◽

Src Homology 3 ◽

Filament Assembly ◽

Tightly Coupled ◽

Src Homology 3 Domain ◽

Src Homology

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

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Cell Shape Related Ultrastructural Changes of Monkey Luteal Cells in Culture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003068 ◽

1980 ◽

Vol 38 ◽

pp. 700-701

Author(s):

Bela J. Gulyas

Keyword(s):

Menstrual Cycle ◽

Cell Shape ◽

Culture Medium ◽

Calf Serum ◽

Ultrastructural Changes ◽

Cover Glass ◽

Luteal Cells ◽

Cellular Processes ◽

Cells In Culture

The cellular processes that participate either in the maintenance or the danise of the corpus luteum of the menstrual cycle are incompletely under¬stood. Maintaining dissociated luteal cells in culture enabled us to study some of these regulatory mechanisms in the absence of the complex in vivo milieu. Here, we report SEM and TEM observations on cell shape related cytoplasmic changes of monkey luteal cells maintained in culture. The CL were obtained at mid luteal phase of the menstrual cycle of rhesus monkeys and were dissociated in col1agenase. Dispersed luteal cells were cultured in Costar dishes, with or without cover glass. The culture medium, Ham's FlO and 10% fetal calf serum, was supplemented with 100 ng/ml hCG in half of the cultures.

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Cross-linkers both drive and brake cytoskeletal remodeling and furrowing in cytokinesis

Molecular Biology of the Cell ◽

10.1091/mbc.e17-06-0392 ◽

2018 ◽

Vol 29 (5) ◽

pp. 622-631 ◽

Cited By ~ 28

Author(s):

Carlos Patino Descovich ◽

Daniel B. Cortes ◽

Sean Ryan ◽

Jazmine Nash ◽

Li Zhang ◽

...

Keyword(s):

Cell Shape ◽

Ring Closure ◽

Cytoskeletal Remodeling ◽

Contraction Speed ◽

Nonmuscle Myosin Ii ◽

Nonmuscle Cells ◽

Myosin Motors ◽

Shape Changes ◽

Partial Depletion

Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling.

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The Drosophila Ral GTPase Regulates Developmental Cell Shape Changes through the Jun NH2-terminal Kinase Pathway

The Journal of Cell Biology ◽

10.1083/jcb.146.2.361 ◽

1999 ◽

Vol 146 (2) ◽

pp. 361-372 ◽

Cited By ~ 39

Author(s):

Kazunobu Sawamoto ◽

Per Winge ◽

Shinya Koyama ◽

Yuki Hirota ◽

Chiharu Yamada ◽

...

Keyword(s):

Cell Shape ◽

Effector Proteins ◽

Dominant Negative ◽

Loss Of Function ◽

Jnk Pathway ◽

Cell Shape Changes ◽

Ral Gtpase ◽

Shape Changes ◽

Constitutively Active

The Ral GTPase is activated by RalGDS, which is one of the effector proteins for Ras. Previous studies have suggested that Ral might function to regulate the cytoskeleton; however, its in vivo function is unknown. We have identified a Drosophila homologue of Ral that is widely expressed during embryogenesis and imaginal disc development. Two mutant Drosophila Ral (DRal) proteins, DRalG20V and DRalS25N, were generated and analyzed for nucleotide binding and GTPase activity. The biochemical analyses demonstrated that DRalG20V and DRalS25N act as constitutively active and dominant negative mutants, respectively. Overexpression of the wild-type DRal did not cause any visible phenotype, whereas DRalG20V and DRalS25N mutants caused defects in the development of various tissues including the cuticular surface, which is covered by parallel arrays of polarized structures such as hairs and sensory bristles. The dominant negative DRal protein caused defects in the development of hairs and bristles. These phenotypes were genetically suppressed by loss of function mutations of hemipterous and basket, encoding Drosophila Jun NH2-terminal kinase kinase (JNKK) and Jun NH2-terminal kinase (JNK), respectively. Expression of the constitutively active DRal protein caused defects in the process of dorsal closure during embryogenesis and inhibited the phosphorylation of JNK in cultured S2 cells. These results indicate that DRal regulates developmental cell shape changes through the JNK pathway.

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The Epithelial-to-Mesenchymal Transition in Development and Cancer

Annual Review of Cancer Biology ◽

10.1146/annurev-cancerbio-030518-055425 ◽

2020 ◽

Vol 4 (1) ◽

pp. 197-220 ◽

Cited By ~ 2

Author(s):

Alexandre Francou ◽

Kathryn V. Anderson

Keyword(s):

Cancer Progression ◽

Cell Shape ◽

Epithelial To Mesenchymal Transition ◽

Dynamic Imaging ◽

Molecular Regulation ◽

Mesenchymal Transition ◽

Cellular Processes ◽

Cellular Events ◽

Neural Crest Development

Epithelial-to-mesenchymal transitions (EMTs) are complex cellular processes where cells undergo dramatic changes in signaling, transcriptional programming, and cell shape, while directing the exit of cells from the epithelium and promoting migratory properties of the resulting mesenchyme. EMTs are essential for morphogenesis during development and are also a critical step in cancer progression and metastasis formation. Here we provide an overview of the molecular regulation of the EMT process during embryo development, focusing on chick and mouse gastrulation and neural crest development. We go on to describe how EMT regulators participate in the progression of pancreatic and breast cancer in mouse models, and discuss the parallels with developmental EMTs and how these help to understand cancer EMTs. We also highlight the differences between EMTs in tumor and in development to arrive at a broader view of cancer EMT. We conclude by discussing how further advances in the field will rely on in vivo dynamic imaging of the cellular events of EMT.

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Rac1 Is Crucial for Hair Follicle Integrity but Is Not Essential for Maintenance of the Epidermis

Molecular and Cellular Biology ◽

10.1128/mcb.00075-06 ◽

2006 ◽

Vol 26 (18) ◽

pp. 6957-6970 ◽

Cited By ~ 87

Author(s):

Anna Chrostek ◽

Xunwei Wu ◽

Fabio Quondamatteo ◽

Rong Hu ◽

Anna Sanecka ◽

...

Keyword(s):

Hair Follicle ◽

Hair Follicles ◽

Small Gtpase ◽

Epidermal Keratinocytes ◽

Sebaceous Glands ◽

Cellular Processes ◽

Normal Differentiation ◽

Deficient Mice

ABSTRACT Rac1 is a small GTPase that regulates the actin cytoskeleton but also other cellular processes. To investigate the function of Rac1 in skin, we generated mice with a keratinocyte-restricted deletion of the rac1 gene. Rac1-deficient mice lost nearly all of their hair within a few weeks after birth. The nonpermanent part of mutant hair follicles developed constrictions; lost expression of hair follicle-specific keratins, E-cadherin, and α6 integrin; and was eventually removed by macrophages. The permanent part of hair follicles and the sebaceous glands were maintained, but no regrowth of full-length hair follicles was observed. In the skin of mutant mice, epidermal keratinocytes showed normal differentiation, proliferation, cell-cell contacts, and basement membrane deposition, demonstrating no obvious defects of Rac1-deficient epidermis in vivo. In vitro, Rac1-null keratinocytes displayed a strong spreading defect and slightly impaired adhesion. These data show that Rac1 plays an important role in sustaining the integrity of the lower part of hair follicles but not in maintenance of the epidermis.

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