Shear Force at the Cell-Matrix Interface: Enhanced Analysis for Microfabricated Post Array Detectors

The interplay of mechanical forces between the extracellular environment and the cytoskeleton drives development, repair, and senescence in many tissues. Quantitative definition of these forces is a vital step in understanding cellular mechanosensing. Microfabricated post array detectors (mPADs) provide direct measurements of cell-generated forces during cell adhesion to extracellular matrix. A new approach to mPAD post labeling, volumetric imaging, and an analysis of post bending mechanics determined that cells apply shear forces and not point moments at the matrix interface. In addition, these forces could be accurately resolved from post deflections by using images of post tops and bases. Image analysis tools were then developed to increase the precision and throughput of post centroid location. These studies resulted in an improved method of force measurement with broad applicability and concise execution using a fully automated force analysis system. The new method measures cell-generated forces with less than 5% error and less than 90 seconds of computational time. Using this approach, we demonstrated direct and distinct relationships between cellular traction force and spread cell surface area for fibroblasts, endothelial cells, epithelial cells and smooth muscle cells.     

Optogenetic fUSI for brain-wide mapping of neural activity mediating collicular-dependent behaviors

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Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta)

Summary Studies of monoplastidic mitosis in hornworts (Bryophyta) using transmission electron microscopy and indirect immunofluorescence staining of microtubules have revealed that two mutually perpendicular microtubule systems predict division polarity in preprophase. Events of cytoplasmic reorganization in preparation for division occur in the following order: migration of the single plastid to a position perpendicular to the division site, constriction of the plastid where its midpoint intersects the division site, development of an axial system of microtubules parallel to the elongating plastid isthmus, and appearance of an atypical preprophase band of microtubules (PPB). The PPB is asymmetrical with a tight band of microtubules on the side over the plastid isthmus and a broad band of widely spaced microtubules over the nucleus. The axial system contributes directly to development of the spindle. In prometaphase, the axial system separates at the equator and additional microtubule bundles project from polar regions, creating two opposing halfspindles. The PPB is still present during asymmetrical organization of the spindle and microtubules extending from the broad portion of the PPB to poles appear to be incorporated into the developing spindle. Dynamic changes in the microtubular cytoskeleton demonstrate (1) intimate relationship of plastid and nuclear division, (2) contribution of preprophase/prophase microtubule systems to spindle development in monoplastidic cells, and (3) dynamic reorientation of microtubules from one system to another.     

Pollen development in orchids 4. Cytoskeleton and ultrastructure of the unequal pollen mitosis inPhalaenopsis

Summary The unequal first mitosis in pollen ofPhalaenopsis results in a small generative cell cut off at the distal surface of the microspore and a large vegetative cell. No preprophase band of microtubules is present, but polarization of the microspore prior to this critical division is well marked. A generative pole microtubule system (GPMS) marks the path of nuclear migration to the distal surface, and the organelles become unequally distributed. Mitochondria, plastids and dictyosomes are concentrated around the vegetative pole in the center of the microspore and are almost totally excluded from the generative pole. The prophase spindle is multipolar with a dominant convergence center at the GPMS site. The metaphase spindle is disc-shaped with numerous minipoles terminating in broad polar regions. In anaphase, the spindle becomes cone-shaped as the spindle elongates and the vegetative pole narrows. These changes in spindle architecture are reflected in the initial shaping of the telophase chromosome groups. F-actin is coaligned with microtubules in the spindle and is also seen as a network in the cytoplasm. An outstanding feature of orchid pollen mitosis is the abundance of endoplasmic reticulum (ER) associated with the spindle. ER extends along the kinetochore fibers, and the numerous foci of spindle fibers at the broad poles terminate in a complex of ER.Abbreviations CLSM confocal laser scanning microscope/microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - GPMS generative pole microtubule system - MBS m-maleimidobenzoic acidN-hydroxysuccinimide ester - PPB preprophase band of microtubules - RhPh rhodamine palloidin - TEM transmission electron microscope/microscopy     

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

ErbB3/HER3 does not homodimerize upon neuregulin binding at the cell surface

To understand signaling by the neuregulin (NRG) receptor ErbB3/HER3, it is important to know whether ErbB3 forms homodimers upon ligand binding. Previous biophysical studies suggest that the ErbB3 extracellular region remains monomeric when bound to NRG. We used a chimeric receptor approach to address this question in living cells, fusing the extracellular region of ErbB3 to the kinase-active intracellular domain of ErbB1. The ErbB3/ErbB1 chimera responded to NRG only if ErbB2 was co-expressed in the same cells, whereas an ErbB4/ErbB1 chimera responded without ErbB2. We, therefore, suggest that ErbB3 is an obligate heterodimerization partner because of its inability to homodimerize.