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The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryoelectron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin. [ABSTRACT FROM AUTHOR]

Drosophila is a powerful model in which to perform genetic screens, but screening assays that are both rapid and can be used to examine a wide variety of cellular and molecular pathways are limited. Drosophila offer an extensive toolbox of GFP-based transcriptional reporters, GFP-tagged proteins, and driver lines, which can be used to express GFP in numerous subpopulations of cells. Thus, a tool that can rapidly and quantitatively evaluate GFP levels in Drosophila tissue would provide a broadly applicable screening platform. We developed a GFP-based enzyme-linked immunosorbent assay (ELISA) that can detect GFP in Drosophila lysates collected from whole animals and dissected tissues across all stages of Drosophila development. We demonstrate that this assay can detect membrane-localized GFP in a variety of neuronal and glial populations and validate that it can identify genes that change the morphology of these cells, as well as changes in STAT and JNK transcriptional activity. We found that this assay can detect endogenously GFP-tagged proteins, including Draper, Cryptochrome, and the synaptic marker Brp. This approach is able to detect changes in Brp-GFP signal during developmental synaptic remodeling, and known genetic regulators of glial synaptic engulfment could be identified using this ELISA method. Finally, we used the assay to perform a small-scale screen, which identified Syntaxins as potential regulators of astrocyte-mediated synapse elimination. Together, these studies establish an ELISA as a rapid, easy, and quantitative in vivo screening method that can be used to assay a wide breadth of fundamental biological questions. [ABSTRACT FROM AUTHOR]

During growth, cells must expand their cell volumes in coordination with biomass to control the level of cytoplasmic macromolecular crowding. Dry-mass density, the average ratio of dry mass to volume, is roughly constant between different nutrient conditions in bacteria, but it remains unknown whether cells maintain dry-mass density constant at the single-cell level and during nonsteady conditions. Furthermore, the regulation of dry-mass density is fundamentally not understood in any organism. Using quantitative phase microscopy and an advanced image-analysis pipeline, we measured absolute single-cell mass and shape of the model organisms Escherichia coli and Caulobacter crescentus with improved precision and accuracy. We found that cells control dry-mass density indirectly by expanding their surface, rather than volume, in direct proportion to biomass growth--according to an empirical surface growth law. At the same time, cell width is controlled independently. Therefore, cellular dry-mass density varies systematically with cell shape, both during the cell cycle or after nutrient shifts, while the surface-to-mass ratio remains nearly constant on the generation time scale. Transient deviations from constancy during nutrient shifts can be reconciled with turgor-pressure variations and the resulting elastic changes in surface area. Finally, we find that plastic changes of cell width after nutrient shifts are likely driven by turgor variations, demonstrating an important regulatory role of mechanical forces for width regulation. In conclusion, turgor-dependent cell width and a slowly varying surface-to-mass coupling constant are the independent variables that determine dry-mass density. [ABSTRACT FROM AUTHOR]

The systematic variation of diameters in branched networks has tantalized biologists since the discovery of da Vinci's rule for trees. Da Vinci's rule can be formulated as a power law with exponent two: The square of the mother branch's diameter is equal to the sum of the squares of those of the daughters. Power laws, with different exponents, have been proposed for branching in circulatory systems (Murray's law with exponent 3) and in neurons (Rall's law with exponent 3/2). The laws have been derived theoretically, based on optimality arguments, but, for the most part, have not been tested rigorously. Using superresolution methods to measure the diameters of dendrites in highly branched Drosophila class IV sensory neurons, we have found that these types of power laws do not hold. In their place, we have discovered a different diameterscaling law: The cross-sectional area is proportional to the number of dendrite tips supported by the branch plus a constant, corresponding to a minimum diameter of the terminal dendrites. The area proportionality accords with a requirement for microtubules to transport materials and nutrients for dendrite tip growth. The minimum diameter may be set by the force, on the order of a few piconewtons, required to bend membrane into the highly curved surfaces of terminal dendrites. Because the observed scaling differs from Rall's law, we propose that cell biological constraints, such as intracellular transport and protrusive forces generated by the cytoskeleton, are important in determining the branched morphology of these cells. [ABSTRACT FROM AUTHOR]

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli’s inner membrane from the cell wall. Shrinkage was accompanied by an ∼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic. [ABSTRACT FROM AUTHOR]

Epithelia have distinct cellular architectures which are established in development, reestablished after wounding, and maintained during tissue homeostasis despite cell turnover and mechanical perturbations. In turn, cell shape also controls tissue function as a regulator of cell differentiation, proliferation, and motility. Here, we investigate cell shape changes in a model epithelial monolayer. After the onset of confluence, cells continue to proliferate and change shape over time, eventually leading to a final architecture characterized by arrested motion and more regular cell shapes. Such monolayer remodeling is robust, with qualitatively similar evolution in cell shape and dynamics observed across disparate perturbations. Here, we quantify differences in monolayer remodeling guided by the active vertex model to identify underlying order parameters controlling epithelial architecture. When monolayers are formed atop an extracellular matrix with varied stiffness, we find the cell density at which motion arrests varies significantly, but the cell shape remains constant, consistent with the onset of tissue rigidity. In contrast, pharmacological perturbations can significantly alter the cell shape at which tissue dynamics are arrested, consistent with varied amounts of active stress within the tissue. Across all experimental conditions, the final cell shape is well correlated to the cell proliferation rate, and cell cycle inhibition immediately arrests cell motility. Finally, we demonstrate cell cycle variation in junctional tension as a source of active stress within the monolayer. Thus, the architecture and mechanics of epithelial tissue can arise from an interplay between cell mechanics and stresses arising from cell cycle dynamics. [ABSTRACT FROM AUTHOR]

Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, these IF head domains interconvert between states of conformational disorder and labile, β-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for β-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and diseasecausing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, β-strand-enriched self-interactions may broadly influence cell morphology. [ABSTRACT FROM AUTHOR]