Viral transport and the cytoskeleton

In the past decade, studies into the way in which intracellular bacterial pathogens hijack and subvert their hosts have provided many important insights into regulation of the actin cytoskeleton and cell motility, in addition to increasing our understanding of the infection process. Viral pathogens, however, may ultimately unlock more cellular secrets as they are even more dependent on their hosts during their life cycle.     

Pulsation and stabilization: Contractile forces that underlie morphogenesis

Embryonic development involves global changes in tissue shape and architecture that are driven by cell shape changes and rearrangements within cohesive cell sheets. Morphogenetic changes at the cell and tissue level require that cells generate forces and that these forces are transmitted between the cells of a coherent tissue. Contractile forces generated by the actin-myosin cytoskeleton are critical for morphogenesis, but the cellular and molecular mechanisms of contraction have been elusive for many cell shape changes and movements. Recent studies that have combined live imaging with computational and biophysical approaches have provided new insights into how contractile forces are generated and coordinated between cells and tissues. In this review, we discuss our current understanding of the mechanical forces that shape cells, tissues, and embryos, emphasizing the different modes of actomyosin contraction that generate various temporal and spatial patterns of force generation.     

Visual circuit development in Drosophila

Fly visual circuits are organized into lattice-like arrays and layers. Recent genetic studies have provided insights into how these reiterated structures are assembled through stepwise processes and how precise connections are established during development. Afferent-derived morphogens, such as Hedgehog, play a key role in organizing the overall structure by inducing and recruiting target neurons and glia. In turn, the target-derived ligand DWnt4 guides Frizzled2-expressing photoreceptor afferents to their proper destination. Photoreceptor afferents select specific synaptic targets by forming adhesive interactions and regulating actin cytoskeleton in growth cones. Target specificity is probably achieved by restricting the expression of adhesive molecules, such as Capricious, to appropriate presynaptic and postsynaptic partners, and by differentially regulating the function of broadly expressed adhesive molecules such as N-cadherin.     

Sensitivity and specificity amplification in signal transduction

Intracellular signal transduction pathways transmit signals from the cell surface to various intracellular destinations, such as cytoskeleton and nucleus through a cascade of protein-protein interactions and activation events, leading to phenotypic changes such as cell proliferation, differentiation, and death. Over the past two decades, numerous signaling proteins and signal transduction pathways have been discovered and characterized. There are two major classes of signaling proteins: phosphoproteins (e.g., mitogen-activated protein kinases) and guanosine triphosphatases (GTPases; e.g., Ras and G proteins). They both function as molecular switches by addition and removal of one or more high-energy phosphate groups. This review discusses developments that seek to quantify the signal transduction processes with kinetic analysis and mathematical modeling of the signaling phosphoproteins and GTPases. These studies have provided insights into the sensitivity and specificity amplification of biological signals in integrated systems.     

Shaping the actin cytoskeleton using microtubule tips

The microtubule cytoskeleton serves as a primary spatial regulator of cell shape. As part of their function, microtubules appear to activate or inhibit the actin cytoskeleton at specific locations at the cell cortex for cell polarization, cell migration and cytokinesis. Recent studies reveal molecular insights into these processes. Regulators of the actin cytoskeleton, such as activators of formin and Rho GTPases, are transported to specific sites on the cortex by riding on the plus ends of microtubules.     

Oncogene cooperation in cellular transformation

Oncogenic cell transformation induces major changes in the structure and physiology of the cells: modifications of morphology, differentiation block, disorganisation of cytoskeleton and extracellular matrix, alterations in growth control. The identification of oncogenes relies upon transfer into host normal cells of DNA isolated from cancer cells. The recent development of DNA transfer into germinal cells has provided new insights into the genetic control of tumorogenesis in vivo. In most cases, full transformation into leukemic or tumor cell requires the cooperation of several oncogenes. These observations support the hypothesis of cancer as a multistep process. However, many of the cooperative oncogenes have not yet been identified, especially in human cancers. The recent discovery of genes acting as repressors of cell growth in normal cells has brought to light a new class of potential recessive oncogenes that might have a contributory function in cancer development.     

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events

The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.     

Surfing pathogens and the lessons learned for actin polymerization

A number of unrelated bacterial species as well as vaccinia virus (ab)use the process of actin polymerization to facilitate and enhance their infection cycle. Studies into the mechanism by which these pathogens hijack and control the actin cytoskeleton have provided many interesting insights into the regulation of actin polymerization in migrating cells. This review focuses on what we have learnt from the actin-based motilities of Listeria, Shigella and vaccinia and discusses what we would still like to learn from our nasty friends, including enteropathogenic Escherichia coli and Rickettsia     

Using transgenic zebrafish (Danio rerio) to study development of the craniofacial skeleton

Transgenic zebrafish provide amazing new tools for following and manipulating cells that form the skeleton. Transgenes that label distinct populations of embryonic cells, such as neural crest that forms most of the skull vault as well as the jaws and gills can be used to determine the embryonic origins of cartilages and bones. This provides a powerful model system for studies of the developmental basis for human birth defects and in a comparative context provides new insights into the developmental changes underlying morphological evolution. Targeting transgenes to nuclear or membrane compartments allows detailed tracking of cell shapes and movements. Here we review how such transgenic markers combined with mutants or tissue grafts to generate mosaic zebrafish embryos have already provided many new insights into skeletal development and disease. In the long run, transgenics designed to perturb gene expression hold great promise for studies of gene function.     

Early events in host-pathogen interactions.

Research focused on early events in host-pathogen interactions has provided new insights into fundamental aspects of microbial pathogenicity and plant responses. Considerable progress has been made in understanding regulation of the delivery of pathogenicity determinants from bacteria into plant cells, signal cascades involved in fungal pathogenicity, the co-ordinating role of the plant cytoskeleton in plant defence and calcium flux as a primary signalling function during the hypersensitive reaction.     

Bending over backwards: BAR proteins and the actin cytoskeleton in mammalian receptor-mediated endocytosis

The role of the actin cytoskeleton during receptor-mediated endocytosis (RME) has been well characterized in yeast for many years. Only more recently has the interplay between the actin cytoskeleton and RME been extensively explored in mammalian cells. These studies have revealed the central roles of BAR proteins in RME, and have demonstrated significant roles of BAR proteins in linking the actin cytoskeleton to this cellular process. The actin cytoskeleton generates and transmits mechanical force to promote the extension of receptor-bound endocytic vesicles into the cell. Many adaptor proteins link and regulate the actin cytoskeleton at the sites of endocytosis. This review will cover key effectors, adaptors and signalling molecules that help to facilitate the invagination of the cell membrane during receptor-mediated endocytosis, including recent insights gained on the roles of BAR proteins. The final part of this review will explore associations of alterations to genes encoding BAR proteins with cancer.     

How do cells move along surfaces?

The movement of cells along surfaces is a complex phenomenon that consists of several interrelated processes, including cell-substratum adhesion, and extension and retraction of the cell edge, in which the actin cytoskeleton plays a crucial role. The past decade has seen increasingly detailed molecular-based investigations into cell motility, but it is still not known how molecular events are integrated to give cell movement. Molecular studies are now beginning to be linked to a more global concept of how whole cells move, and this combined approach promises to yield new insights into cell locomotion.     

Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation

Stem cells have been shown to have the potential to provide a source of cells for applications to tissue engineering and organ repair. The mechanisms that regulate stem cell fate, however, mostly remain unclear. Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are isolated from bone marrow and other adult tissues, and can be differentiated into multiple cell lineages, such as bone, cartilage, fat, muscles and neurons. Although previous studies have focused intensively on the effects of chemical signals that regulate MSC commitment, the effects of physical/mechanical cues of the microenvironment on MSC fate determination have long been neglected. However, several studies provided evidence that mechanical signals, both direct and indirect, played important roles in regulating a stem cell fate. In this review, we summarize a number of recent studies on how cell adhesion and mechanical cues influence the differentiation of MSCs into specific lineages. Understanding how chemical and mechanical cues in the microenvironment orchestrate stem cell differentiation may provide new insights into ways to improve our techniques in cell therapy and organ repair.     

Development and application of probes for labeling the actin cytoskeleton in living plant cells

The actin cytoskeleton is one of the most important components of eukaryotic cytoskeletons. It participates in numerous crucial procedures of cells and has been studied by using various methods. The development and application of appropriate probes for actin visualization is the first and foremost step for functional analysis of actin in vivo. Since the actin cytoskeleton is a highly dynamic and sensitive structure, methods previously used to visualize actin often harm cells and cannot reveal the native state of the actin cytoskeleton in living cells. The development of labeling technologies for living plant cells, especially the emergence and application of green fluorescent protein-tagged actin markers, has provided new insights into the structure and function of the actin cytoskeleton in vivo. There has been a number of probes for actin labeling in living plant cells though they each present different advantages and defects. In this review, we discuss and compare those widely used methods for actin visualization and analysis.     

Cytoskeletal reorganization in skeletal muscle differentiation: from cell morphology to gene expression

Actin cytoskeleton profoundly influence a variety of signaling events, including those related to cell growth, survival and differentiation. Recent evidence have provided insights into the mechanisms underlying the ability of cytoskeleton to regulate signal transduction cascades involved in muscle development. This review will deal with the most recent aspects of this field paying particular attention to the role played by actin dynamics in the induction of skeletal muscle-specific genes.     

Atrophy and programmed cell death of skeletal muscle

Striated skeletal is subject to nonlethal cycles of atrophy in response to a variety of physiological and pathological stimuli, including: starvation, disuse, denervation and inflammation. These cells can also undergo cell death in response to appropriate developmental signals or specific pathological insults. Most of the insights gained into the control of vertebrate skeletal muscle atrophy and death have resulted from experimental interventions rather than natural processes. In contrast, the intersegmental muscles (ISMs) of moths are giant cells that initiate sequential and distinct programs of atrophy and death at the end of metamorphosis as a normal component of development. This model has provided fundamental information about the control, biochemistry, molecular biology and anatomy of naturally occurring atrophy and death in vivo. The ISMs have provided a good complement to studies in vertebrates and may provide insights into clinically relevant disorders.     

Ornithine decarboxylase regulates the activity and localization of rhoA via polyamination

Ornithine decarboxylase (ODC) is the rate-limiting enzyme of polyamine synthesis. Polyamines and ODC are connected to cell proliferation and transformation. Resting cells display a low ODC activity while normal, proliferating cells display fluctuations in ODC activity that coincide with changes in the actin cytoskeleton during the cell cycle. Cancerous cells display constitutively elevated ODC activity. Overexpression of ODC in NIH 3T3 fibroblasts induces a transformed phenotype. The cytoskeletal rearrangements during cytokinesis and cell transformation are intimately coupled to the ODC activity but the molecular mechanisms have remained elusive.In this study we investigated how ODC and polyamines influence the organization of the cytoskeleton. Given that the small G-proteins of the rho family are key modulators of the actin cytoskeleton, we investigated the molecular interactions of rhoA with ODC and polyamines. Our results show that transglutaminase-catalyzed polyamination of rhoA regulates its activity. The polyamination status of rhoA crucially influences the progress of the cell cycle as well as the rate of transformation of rat fibroblasts infected with temperature-sensitive v-src. We also show that ODC influences the intracellular distribution of rhoA. These findings provide novel insights into the mechanisms by which ODC and polyamines regulate the dynamics of the cytoskeleton during cell proliferation and transformation.     

Regulation of innate immunity by Rho GTPases

Leukocytes are key cellular components of innate immunity. These phagocytic cells respond to bacteria at sites of infection through chemotactic sensing and directed motility regulated by Rho GTPases. The development of sensitive probes of Rho GTPase dynamics has provided insights into the temporal and spatial aspects of GTPase regulation during chemotaxis and subsequent microbial phagocytosis. The resulting destruction of ingested bacteria by means of reactive oxygen species (ROS) depends on a Rac-regulated "molecular switch" that is modulated by antagonistic crosstalk involving Cdc42. Recent studies of leukocytes derived from Rac1- and Rac2-knockout mice have shown that these highly homologous GTPases have unique biological roles. An understanding of the biochemical basis for such distinct activities should provide novel insights into the molecular details of Rho GTPase function and regulation in innate immunity.