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Cell division in plant cells requires the deposition of a new cell wall between the two daughter cells. The assembly of this plate requires the coordinated movement of cargo vesicles whose size is below the diffraction-limited resolution of the optical microscope. We combined high spatial and temporal resolution confocal laser scanning microscopy with advanced image-processing tools and fluorescence fluctuation methods and distinguished three distinct phases during cell plate expansion in tobacco (Nicotiana tabacum) 'Bright Yellow-2' cells: massive delivery of preexisting vesicles to a disk-shaped region at the equatorial plane precedes a primary rapid expansion phase followed by a secondary, slow expansion phase during which the extremity of the circular plate seeks contact with the mother wall and brings about the separation of the two portions of cytoplasm. Different effects of pharmacological inhibition emphasize the distinct nature of the assembly and expansion mechanisms characterizing these phases.

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area., Podosomes are adhesive cytoskeletal structures found in several cell types, but whether or how they are interconnected is not known. Here the authors demonstrate mesoscale connectivity of podosome clusters by imaging directional flow patterns of podosome components vinculin, talin and F-actin.

Resume Le travail de soins en secteur psychiatrique et particulierement l’accueil des urgences psychiatriques en Centre hospitalier general se confrontent regulierement a des situations de decompensations psychiques aigues liees a des situations de travail. Il peut s’agir de crises psychiques avec risque de passage a l’acte suicidaire, hetero-agressif ou clastique, de syndromes psychotraumatiques ou encore de psychoses aigues. Cette experience clinique amene a s’interroger sur la notion d’urgence en psychopathologie du travail et sur les modalites d’interventions les plus adaptees pour faire face a la necessite d’une action therapeutique immediate et specialisee sans pour autant occulter la place du travail dans le processus de decompensation psychique.

Hsp27 and alphaB-crystallin are molecular chaperones that are constitutively expressed in several mammalian cells, particularly in pathological conditions. These proteins share functions as diverse as protection against toxicity mediated by aberrantly folded proteins or oxidative-inflammation conditions. In addition, these proteins share anti-apoptotic properties and are tumorigenic when expressed in cancer cells. This review summarizes the current knowledge about Hsp27 and alphaB-crystallin and the implications, either positive or deleterious, of these proteins in pathologies such as neurodegenerative diseases, myopathies, asthma, cataracts and cancers. Approaches towards therapeutic strategies aimed at modulating the expression and/or the activities of Hsp27 and alphaB-crystallin are presented.

Current techniques to acquire patient surface data are often very expensive and lack flexibility. In this study, the use of the Microsoft Kinect to reliably acquire 3D scans of patient surface is investigated. A design is presented to make the system easily applicable to the clinic. Potential applications of the device to radiotherapy are also presented. Scan reproducibility was tested by repeatedly scanning an anthropomorphic phantom. Scan accuracy was tested by comparing Kinect scans to the surface extracted from a CT dataset of a Rando® anthropomorphic phantom, which was considered as the true reference surface. Average signed distances of 0.12 ± 2.34 mm and 0.13 ± 2.04 mm were obtained between the compared surfaces for reproducibility and accuracy respectively. This is conclusive, since it indicates that the variations observed come largely from noise distributed around an average distance close to 0 mm. Moreover, the range of the noise is small enough for the system to reliably capture a patient's surface. A system was also designed using two Kinects used together to acquire 3D surfaces in a quick and stable way that is applicable to the clinic. Finally, applications of the device to radiotherapy are demonstrated. Its use to detect localmore » positioning errors is presented, where small local variations difficult to see with the naked eye are clearly visible. The system was also used to predict collisions using gantry and patient scans and thus ensure the safety of unconventional trajectories.« less

Cell migration is a complex process that involves an intricate choreography between cytoskeleton and adhesion molecules with precise regulation of their transport and interactions in space and time in living cells. Although there have been many advances recently in improving spatial resolution of optical microscopy methods, temporal resolution remains a bottleneck for many methods. Previously we have reported spatio-temporal image cross-correlation spectroscopy (STICCS) in combination with TIRF microscopy to provide snap shots of flow fields of adhesion proteins and the cyctoskeleton in migrating cells. However, the temporal resolution of the STICCS transport maps was low compared to the frame time resolution of the TIRF imaging. Here we report the extension of STICCS to its absolute temporal resolution limit as set by the imaging frame rate by applying a short correlation window of 10 or fewer frames with the analysis window iterated sequentially by single frame steps. Although computationally more intensive, this enables us to capture the time evolution of the flow transport and interactions of adhesion components in living cells with a STICCS vector map for every frame of the TIRF movie. We illustrate the method with measurements of time evolving transport maps of the adhesion related macromolecules alpha5, alpha6 and alphaL integrins with paxillin, and actin within, or associated with the basal membrane in adherent U2OS and CHO.B2 cells plated on extracellular matrix components fibronectin, laminin, or ICAM-1. The time resolved cross-correlation vector maps clearly show that the dynamic interactions between alpha6 or alphaL integrins with paxillin evolve in space and time only at active adhesions in protruding and retracting regions of the cells.

Extensive literature is available on how focal adhesions sense and respond to environmental cues during fast migration in polarized cells. In contrast, little is known about how cells feel and translate environmental cues during slow mesenchymal migration through tissues (e.g. dendritic cells, macrophages). These cells form many more podosomes than focal adhesions and use them as mechanosensors to probe and remodel the extracellular matrix. Each podosome consists of a dense, protrusive actin core and an adhesive ring of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Podosomes are highly dynamic, and their actin content continuously fluctuates mediating their protruding activity. Podosomes are organized in large clusters. Electron microscopy showed that podosomes are interconnected by cytoskeletal fibers, suggesting the existence of a mesoscale organization. By combining image correlation spectroscopy (ICS) techniques such as raster ICS (RICS) and time-resolved spatiotemporal ICS (trSTICS), we investigated the collective dynamic behavior of podosome clusters. We compared the dynamic behavior of the mechanosensitive protein vinculin and the mechano-insensitive protein talin. RICS showed similar diffusion (D=2.4 and 2.0μm2/s) and binding (t=0.024 and 0.026s) properties for vinculin and talin. In contrast trSTICS revealed significant differences in their flow patterns. While waves of correlated flow, with speeds ranging from 0.01–0.16 μm/min, are visible throughout the podosome cluster for vinculin, talin dynamics show no clear directionality. Moreover, podosome formation and dissolution are accompanied by characteristic flow patterns of vinculin, suggesting that localized recruitment of mechanosensitive proteins could coordinate podosome protrusive forces. Finally, using cross correlation STICS we show that vinculin, but not talin, dynamics correlate to flow patterns in the actin network. Taken together, our data demonstrate coordinated behavior of mechanosensitive adhesion proteins in podosome clusters providing evidence for mesoscale coordinated protrusive dynamics.

The delivery of new cell wall material to the forming cell plate of a dividing plant cell requires intricate coordination of secretory vesicle trafficking. The vesicles need to be transported rapidly and efficiently to precise locations in the cell at specific times in order for cell division to occur normally. The trafficking of vesicles is mediated by the cytoskeleton via complex regulatory mechanisms. However, the dynamics of the vesicle delivery is difficult to measure in living cells due to their small size and high density. The vesicle dynamics are measureable via Spatio-Temporal Image Correlation Spectroscopy (STICS), a fluorescence fluctuation method that was initially developed to measure the directed transport or flow of proteins inside living cells. STICS relies on calculating the complete space-time correlation function of the intensity fluctuations between images of a time series obtained using a fluorescence microscope. Here, we use STICS to analyze laser scanning confocal microscopy image time series to obtain quantitative information on secretory vesicle dynamics in plant cells between their production from Golgi stacks and the final step of vesicle docking and fusion at the cell plate initiation site. We were able to map the direction and magnitude of vesicle movement at the different stages of cell division. This allowed us to determine the range of velocities of vesicles and to observe the varying flow patterns and the fast changing nature of their dynamics during the formation of the cell division plate.

In plants, cellular growth requires the assembly of extensive amounts of new cell wall surface. The targeted deposition of building material through exocytosis - cell wall polymers, enzymes and membrane material - is therefore a crucial regulatory feature in plant development. The spatial and temporal regulation of the delivery of cargo vesicles to the target surfaces are poorly understood. Spatio-temporal image correlation spectroscopy (STICS) was used to quantify the intracellular dynamics of secretory vesicles and of the actin arrays in pollen tubes - rapidly and polarly growing plant cells. The dynamic profiles were used to validate mathematical models for vesicular trafficking. Boundary conditions were the expanding cell wall (Fig.1A) and the actin array whose shape was obtained by imposing a steady state and constant polymerization rate of the actin filaments (Fig.1B). The model correctly predicted the vesicle flow patterns in different types of pollen tubes and provides an explanation for flow dynamics in cellular regions devoid of actin cytoskeleton. It will serve as a basis for understanding how pollen tubes are able to regulate their morphogenetic pattern, for example when responding to a directional trigger by changing the growth direction (Fig.1C).View Large Image | View Hi-Res Image | Download PowerPoint Slide