The shape and differentiation of human mesenchymal stem cells is especially sensitive to the rigidity of their environment; the physical mechanisms involved are unknown. A theoretical model and experiments demonstrate here that the polarization/alignment of stress-fibers within stem cells is a non-monotonic function of matrix rigidity. We treat the cell as an active elastic inclusion in a surrounding matrix whose polarizability, unlike dead matter, depends on the feedback of cellular forces that develop in response to matrix stresses. The theory correctly predicts the monotonic increase of the cellular forces with the matrix rigidity and the alignment of stress-fibers parallel to the long axis of cells. We show that the anisotropy of this alignment depends non-monotonically on matrix rigidity and demonstrate it experimentally by quantifying the orientational distribution of stress-fibers in stem cells. These findings offer a first physical insight for the dependence of stem cell differentiation on tissue elasticity.

Blood clots and thrombi consist primarily of a mesh of branched fibers made of the protein fibrin. We propose a molecular basis for the marked extensibility and negative compressibility of fibrin gels based on the structural and mechanical properties of clots at the network, fiber, and molecular levels. The force required to stretch a clot initially rises linearly and is accompanied by a dramatic decrease in clot volume and a peak in compressibility. These macroscopic transitions are accompanied by fiber alignment and bundling after forced protein unfolding. Constitutive models are developed to integrate observations at spatial scales that span six orders of magnitude and indicate that gel extensibility and expulsion of water are both manifestations of protein unfolding, which is not apparent in other matrix proteins such as collagen.Fibrin clots are proteinaceous gels that polymerize in the blood as a consequence of biochemical cascades at sites of vascular injury. Together with platelets, this meshwork stops bleeding and supports active contraction during wound healing (1,2). Fibrin also provides a scaffold for thrombi, clots that block blood vessels and cause tissue damage, leading to myocardial infarction, ischemic stroke, and other cardiovascular diseases (3). To maintain hemostasis while minimizing the impact of thrombosis, fibrin must have suitable stiffness and plasticity (4), but also sufficient permeability so that the network can be effectively decomposed (lysed) by proteolytic enzymes (5,6). It is challenging to meet all of these conditions because open scaffolds would be expected to break at low strains, as is true for collagen gels (7). To address how fibrin clots are both permeable and highly extensible, we studied fibrin structures across multiple spatial scales, from whole clots to single fibers and single molecules (Fig. 1).Fibrin clots were made from purified human fibrinogen under conditions (8) that resulted in the formation of long, straight fibers, similar to those found in physiological clots. To simplify the interpretation, the clots were covalently ligated with the use of a transglutaminase (blood clotting factor XIIIa), as naturally occurs in the blood, which prevents protofibrils from sliding past one another, thus eliminating persistent creep (9).Measurements of the extensibility of 2-mm-diameter fibrin clots ( Fig. 2A) demonstrated that the clots could be stretched to more than three times their relaxed length before breaking, with an average stretch of 2.7 ± 0.15-fold (n =6)(10). This is comparable to the single-fiber extensibility that is observed when a fibrin fiber is laterally stretched with an atomic force microscope (11). Qualitatively, the resulting force-strain curve for fibrin is similar to those observed for rubbers and other materials made from flexible chains (12). However, for fibrin clots, which are made of longer, straighter fibers than the thermally fluctuating polymer chains in rubber, models of rubber-like elasticity predict a branching density that is wro...

Using low-cost automated tracking microscopes, we have generated a behavioral database for 305 C. elegans strains, including 76 mutants with no previously described phenotype. The database consists of 9,203 short videos segmented to extract behavior and morphology features that are available online for further analysis. The database also includes summary statistics for 702 measures with statistical comparisons to wild-type controls so that phenotypes can be identified and understood by users.

Tissue cells lack the ability to see or hear but have evolved mechanisms to feel into their surroundings and sense a collective stiffness. A cell can even sense the effective stiffness of rigid objects that are not in direct cellular contact -like the proverbial princess who feels a pea placed beneath soft mattresses. How deeply a cell feels into a matrix can be measured by assessing cell responses on a controlled series of thin and elastic gels that are affixed to a rigid substrate. Gel elasticity E is readily varied with polymer concentrations of now-standard polyacrylamide hydrogels, but to eliminate wrinkling and detachment of thin gels from an underlying glass coverslip, vinyl groups are bonded to the glass before polymerization. Gel thickness is nominally specified using micron-scale beads that act as spacers, but gels swell after polymerization as measured by z-section, confocal microscopy of fluorescent gels. Atomic force microscopy (AFM) is used to measure E at gel surfaces, employing stresses and strains that are typically generated by cells and yielding values for E that span a broad range of tissue microenvironments. To illustrate cell sensitivities to a series of thin-to-thick gels, the adhesive spreading of mesenchymal stem cells was measured on gel mimics of a very soft tissue (eg. brain, E ~ 1 kPa). Initial results show that cells increasingly respond to the rigidity of an underlying 'hidden' surface starting at about 10-20 µm gel thickness with a characteristic tactile length of less than about 5 µm.

Some natural influenza viruses need only three amino acid substitutions to acquire airborne transmissibility between mammals.

Visible phenotypes based on locomotion and posture have played a critical role in understanding the molecular basis of behavior and development in Caenorhabditis elegans and other model organisms. However, it is not known whether these human-defined features capture the most important aspects of behavior for phenotypic comparison or whether they are sufficient to discover new behaviors. Here we show that four basic shapes, or eigenworms, previously described for wild-type worms, also capture mutant shapes, and that this representation can be used to build a dictionary of repetitive behavioral motifs in an unbiased way. By measuring the distance between each individual's behavior and the elements in the motif dictionary, we create a fingerprint that can be used to compare mutants to wild type and to each other. This analysis has revealed phenotypes not previously detected by real-time observation and has allowed clustering of mutants into related groups. Behavioral motifs provide a compact and intuitive representation of behavioral phenotypes.phenotyping | imaging | ethology | nematode T he study of unconstrained spontaneous behavior is the core of ethology, and it has also made significant contributions to behavioral genetics in model organisms. A powerful approach has been the careful expert observation of mutants to identify those with visible locomotor phenotypes, as demonstrated for many model organisms (1-6). However, as with most manually scored experiments, subjectivity can reduce reproducibility, whereas subtle quantitative changes or those that happen on very short or long time-scales are likely to be missed. Furthermore, manual observations are not scalable, and this has led to a widening gap between our ability to sequence and manipulate genomes and our ability to assess the effects of genetic variation and mutation on behavior.Several recent reports describe systems that begin to address this gap by automatically recording and quantifying spontaneous behavior in animals ranging from worms (7-15) to flies (16-19), fish (20, 21), and mice (22,23). The advantage of these approaches is that they provide a means to quantify movement parameters such as velocity precisely and in some cases to automatically detect predefined behaviors based on a manually annotated training data set. This automated analysis eliminates some of the problems of a purely manual approach, but it still relies on preselected behavioral parameters that may not be optimal for phenotypic comparisons and precludes the discovery of new behaviors that have not already been observed by eye. An alternative approach is to use unsupervised learning, which attempts to use the inherent structure of a data set to identify informative patterns; to do this, we first needed to extract worm postures from movie data and have as compact and complete a representation of worm behavior as possible.

Crystallization processes are in general sensitive to detailed conditions, but our present understanding of underlying mechanisms is insufficient. A crystallizable chain within a diblock copolymer assembly is expected to couple curvature to crystallization and thereby impact rigidity as well as preferred morphology, but the effects on dispersed phases have remained unclear. The hydrophobic polymer polycaprolactone (PCL) is semi-crystalline in bulk (T m = 60°C) and is shown here to generate flexible worm micelles or rigid vesicles in water from several dozen polyethyleneoxide-based diblocks (PEO-PCL). Despite the fact that `worms' have a mean curvature between that of vesicles and spherical micelles, `worms' are seen only within a narrow, process-dependent wedge of morphological phase space that is deep within the vesicle phase. Fluorescence imaging shows worms are predominantly in one of two states -either entirely flexible with dynamic thermal undulations or fully rigid; only a few worms appear rigid at room temperature (T << T m ), indicating suppression of crystallization by both curvature and PCL hydration. Worm rigidification, which depends on molecular weight, is also prevented by copolymerization of caprolactone with just 10% racemic lactide that otherwise has little impact on bulk crystallinity. In contrast to worms, vesicles of PEO-PCL are always rigid and typically leaky. Defects between crystallite domains induce dislocation-roughening with focal leakiness although select PEO-PCL -which classical surfactant arguments would predict make worms -yield vesicles that retain encapsulant and appear smooth, suggesting a gel or glassy state. Hydration in dispersion thus tends to selectively soften high curvature microphases.Keywords block copolymer; worm micelle; polymersome; crystallinity Corresponding: discher@seas.upenn.edu. Supporting Information. Molecular details of all polymers synthesized, calculation of polymer distribution in assemblies, AFM and fluorescence microscopy images, DLS and dye encapsulation studies, movies of worm micelles, persistence length measurement of worm micelles, procedure for measurement of contour lengths, and phase contrast images of OCL (2, 13.5) can be found in Supporting Information. Supporting Information is available free of charge on the internet at

SUMMARY Fibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the γ chain nodules and reversible extension-contraction of the α-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels.