Stress caused by environmental factors evokes dynamic changes in plant phenotypes. In this study, we deciphered simultaneously the reaction of plant growth and chlorophyll fluorescence related parameters using a novel approach which combines existing imaging technologies (GROWSCREEN FLUORO). Three different abiotic stress situations were investigated demonstrating the benefit of this approach to distinguish between effects related to (1) growth, (2) chlorophyll-fluorescence, or (3) both of these aspects of the phenotype. In a drought stress experiment with more than 500 plants, poly(ADP-ribose) polymerase (PARP) deficient lines of Arabidopsis thaliana (L.) Heynh showed increased relative growth rates (RGR) compared with C24 wild-type plants. In chilling stress, growth of PARP and C24 lines decreased rapidly, followed by a decrease in Fv/Fm. Here, PARP-plants showed a more pronounced decrease of Fv/Fm than C24, which can be interpreted as a more efficient strategy for survival in mild chilling stress. Finally, the reaction of Nicotiana tabacum L. to altered spectral composition of the intercepted light was monitored as an example of a moderate stress situation that affects chlorophyll-fluorescence related, but not growth-related parameters. The examples investigated in this study show the capacity for improved plant phenotyping based on an automated and simultaneous evaluation of growth and photosynthesis at high throughput.

SummaryCardiomyocytes are responsible for the permanent blood flow by coordinated heart contractions. This vital function is accomplished over a long period of time with almost the same performance, although heart properties, as its elasticity, change drastically upon aging or as a result of diseases like myocardial infarction. In this paper we have analyzed late rat embryonic heart muscle cells' morphology, sarcomere/costamere formation and force generation patterns on substrates of various elasticities ranging from ∼1 to 500 kPa, which covers physiological and pathological heart stiffnesses. Furthermore, adhesion behaviour, as well as single myofibril/sarcomere contraction patterns, was characterized with high spatial resolution in the range of physiological stiffnesses (15 kPa to 90 kPa). Here, sarcomere units generate an almost stable contraction of ∼4%. On stiffened substrates the contraction amplitude remains stable, which in turn leads to increased force levels allowing cells to adapt almost instantaneously to changing environmental stiffness. Furthermore, our data strongly indicate specific adhesion to flat substrates via both costameric and focal adhesions. The general appearance of the contractile and adhesion apparatus remains almost unaffected by substrate stiffness.

Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and type II keratin subunits. Missense mutations in keratin 5 or keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS) in humans by rendering the keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe generalized EBS, by stable expression in keratin-free keratinocytes. This revealed distinctly different effects on keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with keratin-free keratinocytes. Thus, a keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS therapy approaches.

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

The skin’s epidermis is a multilayered epithelial tissue and the first line of defense against mechanical stress. Its barrier function depends on an integrated assembly and reorganization of cell–matrix and cell–cell junctions in the basal layer and on different intercellular junctions in suprabasal layers. However, how mechanical stress is recognized and which adhesive and cytoskeletal components are involved are poorly understood. Here, we subjected keratinocytes to cyclic stress in the presence or absence of intercellular junctions. Both states not only recognized but also responded to strain by reorienting actin filaments perpendicular to the applied force. Using different keratinocyte mutant strains that altered the mechanical link of the actin cytoskeleton to either cell–matrix or cell–cell junctions, we show that not only focal adhesions but also adherens junctions function as mechanosensitive elements in response to cyclic strain. Loss of paxillin or talin impaired focal adhesion formation and only affected mechanosensitivity in the absence but not presence of intercellular junctions. Further analysis revealed the adherens junction protein α-catenin as a main mechanosensor, with greatest sensitivity conferred on binding to vinculin. Our data reveal a mechanosensitive transition from cell–matrix to cell–cell adhesions on formation of keratinocyte monolayers with vinculin and α-catenin as vital players.

Neuronal mechanobiology plays a vital function in brain development and homeostasis with an essential role in neuronal maturation, pathfinding, and differentiation but is also crucial for understanding brain pathology. In this study, we constructed an in vitro system to assess neuronal responses to cyclic strain as a mechanical signal. The selected strain amplitudes mimicked physiological as well as pathological conditions. By subjecting embryonic neuronal cells to cyclic uniaxial strain we could steer the direction of neuronal outgrowth perpendicular to strain direction for all applied amplitudes. A long-term analysis proved maintained growth direction. Moreover, stretched neurons showed an enhanced length, growth, and formation of nascent side branches with most elevated growth rates subsequent to physiological straining. Application of cyclic strain to already formed neurites identified retraction bulbs with destabilized microtubule structures as spontaneous responses. Importantly, neurons were able to adapt to the mechanical signals without induction of cell death and showed a triggered growth behavior when compared to unstretched neurons. The data suggest that cyclic strain plays a critical role in neuronal development.

Any cell within a tissue is constantly confronted with a variety of mechanical stimuli. Sensing of these diverse stimuli plays an important role in cellular regulation. Besides shear stress, cells of the vascular endothelium are particularly exposed to a permanent cyclic straining originating from the interplay of outwards pushing blood pressure and inwards acting contraction by smooth musculature. Perpendicular alignment of cells as structural adaptation to this condition is a basic prerequisite in order to withstand deformation forces. Here, we combine live cell approaches with immunocytochemical analyses on single cell level to closely elucidate the mechanisms of cytoskeletal realignment to cyclic strain and consolidate orientation analyses of actin fibres, microtubules (MTs) and vimentin. We could show that strain-induced reorientation takes place for all cytoskeletal systems. However, all systems are characterized by their own, specific reorientation time course with actin filaments reorienting first followed by MTs and finally vimentin. Interestingly, in all cases, this reorientation was faster than cell body realignment which argues for an active adaptation mechanism for all cytoskeletal systems. Upon actin destabilization, already smallest alterations in actin kinetics massively hamper cell morphology under strain and therefore overall reorientation. Depolymerization of MTs just slightly influences actin reorientation velocity but strongly affects cell body reorientation.

Keratin intermediate filaments provide mechanical resilience for epithelia. They are nevertheless highly dynamic and turn over continuously, even in sessile keratinocytes. the aim of this study was to characterize and understand how the dynamic behavior of the keratin cytoskeleton is integrated in migrating cells. By imaging human primary keratinocytes producing fluorescent reporters and by using standardized image analysis we detect inward-directed keratin flow with highest rates in the cell periphery. The keratin flow correlates with speed and trajectory of migration. Changes in fibronectincoating density and substrate stiffness induces concordant changes in migration speed and keratin flow. When keratinocytes are pseudo-confined on stripes, migration speed and keratin flow are reduced affecting the latter disproportionately. The regulation of keratin flow is linked to the regulation of actin flow. Local speed and direction of keratin and actin flow are very similar in migrating keratinocytes with keratin flow lagging behind actin flow. Conversely, reduced actin flow in areas of high keratin density indicates an inhibitory function of keratins on actin dynamics. together, we propose that keratins enhance persistence of migration by directing actin dynamics and that the interplay of keratin and actin dynamics is modulated by matrix adhesions.Cell migration is a highly complex process with relevance for physiological and pathological situations such as embryogenesis, wound healing and metastasis 1 . It is induced by chemical and biophysical cues. The cytoskeleton is at the core of generating effective locomotion by enabling successive steps of protrusion at the cell front and by facilitating the contractile events at the cell rear in parallel with the regulation of cell-matrix adhesions 2 . The cytoskeleton is composed of three major components, namely actin filaments, microtubules and intermediate filaments. The role of actin filaments and microtubules in cell migration has been extensively studied. In contrast, the contribution of intermediate filaments and especially of epithelial keratin intermediate filaments is still poorly understood 3 .Keratin filaments are the main class of cytoplasmic intermediate filaments in epithelial cells. They belong to a large multigene family encoded by more than 50 genes. Every keratin filament contains equal amounts of type I (acidic) and type II (basic) monomers. Obligatory heterodimers assemble in an antiparallel manner into non-polar tetramers, which further associate laterally and longitudinally to eventually generate 8-12 nm filaments 4-6 .The effect of keratin expression on migration depends on the keratin isoform, the cell type and the environment 3,7,8 . For example, the knockdown of K8/K18 reduces the invasion capacity of squamous cancer cells 9 but increases collective cell migration of cancer cells 10 . Depletion of the entire type II keratin gene cluster in mouse keratinocytes prevents keratin filament assembly and induces an increase in migration speed and invasiv...