micromanipulation-techniques-allowing-analysis-morphogenetic-dynamics

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Technische Universität Braunschweig

Helmholtz Centre for Infection Research

The heptameric Arp2/3 complex generates branched actin filament networks that drive lamellipodium protrusion, vesicle trafficking and pathogen motility. Distinct variants of the Arp2/3 complex are now shown to have different roles in tuning actin assembly and disassembly, in concert with the prominent actin regulators cortactin and coronin. 

How distinct Arp2/3 complex variants regulate actin filament assembly.

CELL BIOLOGY. Formin' filaments at a faster CLIP.

Salmonella Typhimurium (S. Tm) is a leading cause of diarrhea. The disease is triggered by pathogen invasion into the gut epithelium. Invasion is attributed to the SPI-1 type 3 secretion system (T1). T1 injects effector proteins into epithelial cells and thereby elicits rearrangements of the host cellular actin cytoskeleton and pathogen invasion. The T1 effector proteins SopE, SopB, SopE2 and SipA are contributing to this. However, the host cell factors contributing to invasion are still not completely understood. To address this question comprehensively, we used Hela tissue culture cells, a genome-wide siRNA library, a modified gentamicin protection assay and S. TmSipA, a sopBsopE2sopE mutant which strongly relies on the T1 effector protein SipA to invade host cells. We found that S. TmSipA invasion does not elicit membrane ruffles, nor promote the entry of non-invasive bacteria "in trans". However, SipA-mediated infection involved the SPIRE family of actin nucleators, besides well-established host cell factors (WRC, ARP2/3, RhoGTPases, COPI). Stage-specific follow-up assays and knockout fibroblasts indicated that SPIRE1 and SPIRE2 are involved in different steps of the S. Tm infection process. Whereas SPIRE1 interferes with bacterial binding, SPIRE2 influences intracellular replication of S. Tm. Hence, these two proteins might fulfill non-redundant functions in the pathogen-host interaction. The lack of co-localization hints to a short, direct interaction between S. Tm and SPIRE proteins or to an indirect effect. 

A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion.

The actin cytoskeleton is essential for morphogenesis and virtually all types of cell shape changes. Reorganization is per definition driven by continuous disassembly and re-assembly of actin filaments, controlled by major, ubiquitously operating machines. These are specifically employed by the cell to tune its activities in accordance with respective environmental conditions or to satisfy specific needs.Here we sketch some fundamental signalling pathways established to contribute to the reorganization of specific actin structures at the plasma membrane. Rho-family GTPases are at the core of these pathways, and dissection of their precise contributions to actin reorganization in different cell types and tissues will thus continue to improve our understanding of these important signalling nodes. Furthermore, we will draw your attention to the emerging theme of actin reorganization on intracellular membranes, its functional relation to Rho-GTPase signalling, and its relevance for the exciting phenomenon autophagy.

Signalling Pathways Controlling Cellular Actin Organization.

Most migrating cells extrude their front by the force of actin polymerization. Polymerization requires an initial nucleation step, which is mediated by factors establishing either parallel filaments in the case of filopodia or branched filaments that form the branched lamellipodial network. Branches are considered essential for regular cell motility and are initiated by the Arp2/3 complex, which in turn is activated by nucleation-promoting factors of the WASP and WAVE families. Here we employed rapid amoeboid crawling leukocytes and found that deletion of the WAVE complex eliminated actin branching and thus lamellipodia formation. The cells were left with parallel filaments at the leading edge, which translated, depending on the differentiation status of the cell, into a unipolar pointed cell shape or cells with multiple filopodia. Remarkably, unipolar cells migrated with increased speed and enormous directional persistence, while they were unable to turn towards chemotactic gradients. Cells with multiple filopodia retained chemotactic activity but their migration was progressively impaired with increasing geometrical complexity of the extracellular environment. These findings establish that diversified leading edge protrusions serve as explorative structures while they slow down actual locomotion.

Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes.

Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.

FMNL formins boost lamellipodial force generation.

Cell migration and cell-cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.

Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly.

The Rho-family small GTPase Cdc42 localizes at plasma membrane and Golgi complex and aside from protrusion and migration operates in vesicle trafficking, endo- and exocytosis as well as establishment and/or maintenance of cell polarity. The formin family members FMNL2 and -3 are actin assembly factors established to regulate cell edge protrusion during migration and invasion. Here we report these formins to additionally accumulate and function at the Golgi apparatus. As opposed to lamellipodia, Golgi targeting of these proteins required both their N-terminal myristoylation and the interaction with Cdc42. Moreover, Golgi association of FMNL2 or -3 induced a phalloidin-detectable actin meshwork around the Golgi. Importantly, functional interference with FMNL2/3 formins by RNAi or CRISPR/Cas9-mediated gene deletion invariably induced Golgi fragmentation in different cell lines. Furthermore, absence of these proteins led to enlargement of endosomes as well as defective maturation and/or sorting into late endosomes and lysosomes. In line with Cdc42 - recently established to regulate anterograde transport through the Golgi by cargo sorting and carrier formation - FMNL2/3 depletion also affected anterograde trafficking of VSV-G from the Golgi to the plasma membrane. Our data thus link FMNL2/3 formins to actin assembly-dependent functions of Cdc42 in anterograde transport through the Golgi apparatus.

FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42.

The WAVE regulatory complex (WRC) links upstream Rho-family GTPase signaling to the activation of the ARP2/3 complex in different organisms. WRC-induced and ARP2/3 complex-mediated actin nucleation beneath the plasma membrane is critical for actin assembly in the leading edge to drive efficient cell migration. The WRC is a stable heteropentamer composed of SCAR/WAVE, Abi, Nap, Pir and the small polypeptide Brk1/Hspc300. Functional interference with individual subunits of the complex frequently results in diminished amounts of the remaining polypeptides of the WRC complex, implying the complex to act as molecular entity. However, Abi was also found to associate with mammalian N-WASP, formins, Eps8/SOS1 or VASP, indicating additional functions of individual WRC subunits in eukaryotic cells. To address this issue systematically, we inactivated all WRC subunits, either alone or in combination with VASP in Dictyostelium cells and quantified the protein content of the remaining subunits in respective WRC knockouts. The individual mutants displayed highly differential phenotypes concerning various parameters, including cell morphology, motility, cytokinesis or multicellular development, corroborating the view of additional roles for individual subunits, beyond their established function in WRC-mediated Arp2/3 complex activation. Finally, our data uncover the interaction of the actin polymerase VASP with WRC-embedded Abi to mediate VASP accumulation in cell protrusions, driving efficient cell migration.

Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes.

Cell spreading requires the coupling of actin-driven membrane protrusion and integrin-mediated adhesion to the extracellular matrix. The integrin-activating adaptor protein kindlin-2 plays a central role for cell adhesion and membrane protrusion by directly binding and recruiting paxillin to nascent adhesions. Here, we report that kindlin-2 has a dual role during initial cell spreading: it binds paxillin via the pleckstrin homology and F0 domains to activate Rac1, and it directly associates with the Arp2/3 complex to induce Rac1-mediated membrane protrusions. Consistently, abrogation of kindlin-2 binding to Arp2/3 impairs lamellipodia formation and cell spreading. Our findings identify kindlin-2 as a key protein that couples cell adhesion by activating integrins and the induction of membrane protrusions by activating Rac1 and supplying Rac1 with the Arp2/3 complex.

Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading.

The actin cytoskeleton and associated motor proteins provide the driving forces for establishing the astonishing morphological diversity and dynamics of mammalian cells. Aside from functions in protruding and contracting cell membranes for motility, differentiation or cell division, the actin cytoskeleton provides forces to shape and move intracellular membranes of organelles and vesicles. To establish the many different actin assembly functions required in time and space, actin nucleators are targeted to specific subcellular compartments, thereby restricting the generation of specific actin filament structures to those sites. Recent research has revealed that targeting and activation of actin filament nucleators, elongators and myosin motors are tightly coordinated by conserved protein complexes to orchestrate force generation. In this Cell Science at a Glance article and the accompanying poster, we summarize and discuss the current knowledge on the corresponding protein complexes and their modes of action in actin nucleation, elongation and force generation.

Actin assembly mechanisms at a glance.

Cell migration frequently involves the protrusion of lamellipodial actin networks, the structure and regulation of which have been studied for decades. New work highlights how the geometry of these networks endows cells with the ability to adapt to environmental conditions and load.

Actin Networks: Adapting to Load through Geometry.

Animal cell migration constitutes a complex process involving a multitude of forces generated and maintained by the actin cytoskeleton. Dynamic changes of the cell surface, for instance to effect cell edge protrusion, are at the core of initiating migratory processes, both in tissue culture models and whole animals. Here we sketch different aspects of imaging representative molecular constituents in such actin-driven processes, which power and regulate the polymerisation of actin filaments into bundles and networks, constituting the building blocks of such protrusions. The examples presented illustrate both the diversity of subcellular distributions of distinct molecular components, according to their function, and the complexity of dynamic changes in protrusion size, shape, and/or orientation in 3D. Considering these dynamics helps mechanistically connecting subcellular distributions of molecular machines driving protrusion and migration with their biochemical function.

Imaging the Molecular Machines That Power Cell Migration.

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and descendants of it lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of αSMA, but not of collagen I α1 (col1a1). While loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3 dependent actin polymerization nor cofilin dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction and TGFβ-induced actin polymerisation correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis.

Article	Year
How distinct Arp2/3 complex variants regulate actin filament assembly. Nature cell biology\| PubMed ID: 26693915	2016
CELL BIOLOGY. Formin' filaments at a faster CLIP. Science (New York, N.Y.)\| PubMed ID: 27199403	2016
A Genome-Wide siRNA Screen Implicates Spire1/2 in SipA-Driven Salmonella Typhimurium Host Cell Invasion. PloS one\| PubMed ID: 27627128	2016
Signalling Pathways Controlling Cellular Actin Organization. Handbook of experimental pharmacology\| PubMed ID: 27757765	2017
Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes. Nature cell biology\| PubMed ID: 27775702	2016
FMNL formins boost lamellipodial force generation. Nature communications\| PubMed ID: 28327544	2017
Efficiency of lamellipodia protrusion is determined by the extent of cytosolic actin assembly. Molecular biology of the cell\| PubMed ID: 28331069	2017
FMNL2 and -3 regulate Golgi architecture and anterograde transport downstream of Cdc42. Scientific reports\| PubMed ID: 28852060	2017
Differential functions of WAVE regulatory complex subunits in the regulation of actin-driven processes. European journal of cell biology\| PubMed ID: 28889942	2017
Kindlin-2 recruits paxillin and Arp2/3 to promote membrane protrusions during initial cell spreading. The Journal of cell biology\| PubMed ID: 28912124	2017
Actin assembly mechanisms at a glance. Journal of cell science\| PubMed ID: 29032357	2017
Actin Networks: Adapting to Load through Geometry. Current biology : CB\| PubMed ID: 29207269	2017
Imaging the Molecular Machines That Power Cell Migration. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29526003	2018
RhoA, Rac1 and Cdc42 differentially regulate aSMA and collagen I expression in mesenchymal stem cells. The Journal of biological chemistry\| PubMed ID: 29700112	2018

Klemens Rottner

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