SPIN90 associates with mDia1 and the Arp2/3 complex to regulate cortical actin organization

Cell shape is controlled by the submembranous cortex, an actomyosin network mainly generated by two actin nucleators: the Arp2/3 complex and the formin mDia1. Changes in relative nucleator activity may alter cortical organization, mechanics and cell shape. Here we investigate how nucleation-promoting factors mediate interactions between nucleators. In vitro, the nucleation-promoting factor SPIN90 promotes formation of unbranched filaments by Arp2/3, a process thought to provide the initial filament for generation of dendritic networks. Paradoxically, in cells, SPIN90 appears to favour a formin-dominated cortex. Our in vitro experiments reveal that this feature stems mainly from two mechanisms: efficient recruitment of mDia1 to SPIN90-Arp2/3 nucleated filaments and formation of a ternary SPIN90-Arp2/3-mDia1 complex that greatly enhances filament nucleation. Both mechanisms yield rapidly elongating filaments with mDia1 at their barbed ends and SPIN90-Arp2/3 at their pointed ends. Thus, in networks, SPIN90 lowers branching densities and increases the proportion of long filaments elongated by mDia1

Drosophila EGFR pathway coordinates stem cell proliferation and gut remodeling following infection

<p>Abstract</p> <p>Background</p> <p>Gut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, <it>Erwinia carotovora carotovora 15</it>, induces a massive increase in stem cell proliferation in the gut of <it>Drosophila</it>. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal.</p> <p>Results</p> <p>To understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult <it>Drosophila </it>undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection.</p> <p>Conclusions</p> <p>This study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.</p

Coupling changes in cell shape to chromosome segregation

Animal cells undergo dramatic changes in shape, mechanics and polarity as they progress through the different stages of cell division. These changes begin at mitotic entry, with cell–substrate adhesion remodelling, assembly of a cortical actomyosin network and osmotic swelling, which together enable cells to adopt a near spherical form even when growing in a crowded tissue environment. These shape changes, which probably aid spindle assembly and positioning, are then reversed at mitotic exit to restore the interphase cell morphology. Here, we discuss the dynamics, regulation and function of these processes, and how cell shape changes and sister chromatid segregation are coupled to ensure that the daughter cells generated through division receive their fair inheritance

Cytoskeletal control of B cell responses to antigens.

The actin cytoskeleton is essential for cell mechanics and has increasingly been implicated in the regulation of cell signalling. In B cells, the actin cytoskeleton is extensively coupled to B cell receptor (BCR) signalling pathways, and defects of the actin cytoskeleton can either promote or suppress B cell activation. Recent insights from studies using single-cell imaging and biophysical techniques suggest that actin orchestrates BCR signalling at the plasma membrane through effects on protein diffusion and that it regulates antigen discrimination through the biomechanics of immune synapses. These mechanical functions also have a role in the adaptation of B cell subsets to specialized tasks during antibody responses

Cell cortex composition and homeostasis resolved by integrating proteomics and quantitative imaging.

The cellular actin cortex is the cytoskeletal structure primarily responsible for the control of animal cell shape and as such plays a central role in cell division, migration, and tissue morphogenesis. Due to the lack of experimental systems where the cortex can be investigated independently from other organelles, little is known about its composition, assembly, and homeostasis. Here, we describe novel tools to resolve the composition and regulation of the cortex. We report and validate a protocol for cortex purification based on the separation of cellular blebs. Mass spectrometry analysis of purified cortices provides a first extensive list of cortical components. To assess the function of identified proteins, we design an automated imaging assay for precise quantification of cortical actomyosin assembly dynamics. We show subtle changes in cortex assembly dynamics upon depletion of the identified cortical component profilin. Our widely applicable integrated method paves the way for systems-level investigations of the actomyosin cortex and its regulation during morphogenesis. © 2013 Wiley Periodicals, Inc

Cellular control of cortical actin nucleation.

The contractile actin cortex is a thin layer of actin, myosin, and actin-binding proteins that subtends the membrane of animal cells. The cortex is the main determinant of cell shape and plays a fundamental role in cell division [1-3], migration [4], and tissue morphogenesis [5]. For example, cortex contractility plays a crucial role in amoeboid migration of metastatic cells [6] and during division, where its misregulation can lead to aneuploidy [7]. Despite its importance, our knowledge of the cortex is poor, and even the proteins nucleating it remain unknown, though a number of candidates have been proposed based on indirect evidence [8-15]. Here, we used two independent approaches to identify cortical actin nucleators: a proteomic analysis using cortex-rich isolated blebs, and a localization/small hairpin RNA (shRNA) screen searching for phenotypes with a weakened cortex or altered contractility. This unbiased study revealed that two proteins generated the majority of cortical actin: the formin mDia1 and the Arp2/3 complex. Each nucleator contributed a similar amount of F-actin to the cortex but had very different accumulation kinetics. Electron microscopy examination revealed that each nucleator affected cortical network architecture differently. mDia1 depletion led to failure in division, but Arp2/3 depletion did not. Interestingly, despite not affecting division on its own, Arp2/3 inhibition potentiated the effect of mDia1 depletion. Our findings indicate that the bulk of the actin cortex is nucleated by mDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting the relative contribution of each nucleator

A new method to measure mechanics and dynamic assembly of branched actin networks

We measured mechanical properties and dynamic assembly of actin networks with a new method based on magnetic microscopic cylinders. Dense actin networks are grown from the cylinders' surfaces using the biochemical Arp2/3-machinery at play in the lamellipodium extension and other force-generating processes in the cell. Under a homogenous magnetic field the magnetic cylinders self-assemble into chains in which forces are attractive and depend on the intensity of the magnetic field. We show that these forces, from piconewtons to nanonewtons, are large enough to slow down the assembly of dense actin networks and controlled enough to access to their non linear mechanical responses. Deformations are measured with nanometer-resolution, well below the optical resolution. Self-assembly of the magnetic particles into chains simplifies experiments and allows for parallel measurements. The combination of accuracy and good throughput of measurements results in a method with high potential for cell and cytoskeleton mechanics. Using this method, we observed in particular a strong non linear mechanical behavior of dense branched actin networks at low forces that has not been reported previously

Missing-in-metastasis MIM/MTSS1 promotes actin assembly at intercellular junctions and is required for integrity of kidney epithelia.

MIM/MTSS1 is a tissue-specific regulator of plasma membrane dynamics, whose altered expression levels have been linked to cancer metastasis. MIM deforms phosphoinositide-rich membranes through its I-BAR domain and interacts with actin monomers through its WH2 domain. Recent work proposed that MIM also potentiates Sonic hedgehog (Shh)-induced gene expression. Here, we generated MIM mutant mice and found that full-length MIM protein is dispensable for embryonic development. However, MIM-deficient mice displayed a severe urinary concentration defect caused by compromised integrity of kidney epithelia intercellular junctions, which led to bone abnormalities and end-stage renal failure. In cultured kidney epithelial (MDCK) cells, MIM displayed dynamic localization to adherens junctions, where it promoted Arp2/3-mediated actin filament assembly. This activity was dependent on the ability of MIM to interact with both membranes and actin monomers. Furthermore, results from the mouse model and cell culture experiments suggest that full-length MIM is not crucial for Shh signaling, at least during embryogenesis. Collectively, these data demonstrate that MIM modulates interplay between the actin cytoskeleton and plasma membrane to promote the maintenance of intercellular contacts in kidney epithelia