Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cell cortex.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Biomimetic Approach of Brain Vasculature Rapidly Characterizes Inter- and Intra-Patient Migratory Diversity of Glioblastoma.

Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.

Mechanically induced topological transition of spectrin regulates its distribution in the mammalian cortex.

The cell cortex is a dynamic assembly that ensures cell integrity during passive deformation or active response by adapting cytoskeleton topologies with poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons. Erythrocytes rely on triangular-like lattices of spectrin tetramers, which in neurons are organized in periodic arrays. We exploited Expansion Microscopy to discover that these two distinct topologies can co-exist in other mammalian cells such as fibroblasts. We show through biophysical measurements and computational modeling that spectrin provides coverage of the cortex and, with the intervention of actomyosin, erythroid-like lattices can dynamically transition into condensates resembling neuron-like periodic arrays fenced by actin stress fibers. Spectrin condensates experience lower mechanical stress and turnover despite displaying an extension close to the contour length of the tetramer. Our study sheds light on the adaptive properties of spectrin, which ensures protection of the cortex by undergoing mechanically induced topological transitions.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Protocol to assess human glioma propagating cell migration on linear micropatterns mimicking brain invasion tracks.

Glioblastoma (GBM) cells invade the brain by following linear structures like blood vessel walls and white matter tracts by using specific motility modes. In this protocol, we describe two micropatterning techniques allowing recapitulation of these linear tracks in vitro: micro-contact printing and deep UV photolithography. We also detail how to maintain, transfect, and prepare human glioma propagating cells (hGPCs) for migration assays on linear tracks, followed by image acquisition and analysis, to measure key parameters of their motility. For complete details on the use and execution of this protocol, please refer to Monzo et al. (2016) and Monzo et al. (2021a).

Adaptive mechanoproperties mediated by the formin FMN1 characterize glioblastoma fitness for invasion.

Glioblastoma are heterogeneous tumors composed of highly invasive and highly proliferative clones. Heterogeneity in invasiveness could emerge from discrete biophysical properties linked to specific molecular expression. We identified clones of patient-derived glioma propagating cells that were either highly proliferative or highly invasive and compared their cellular architecture, migratory, and biophysical properties. We discovered that invasiveness was linked to cellular fitness. The most invasive cells were stiffer, developed higher mechanical forces on the substrate, and moved stochastically. The mechano-chemical-induced expression of the formin FMN1 conferred invasive strength that was confirmed in patient samples. Moreover, FMN1 expression was also linked to motility in other cancer and normal cell lines, and its ectopic expression increased fitness parameters. Mechanistically, FMN1 acts from the microtubule lattice and promotes a robust mechanical cohesion, leading to highly invasive motility.

Nuclear morphometrics and chromatin condensation patterns as disease biomarkers using a mobile microscope.

Current cancer diagnosis involves the use of nuclear morphology and chromatin condensation signatures for accurate advanced stage classification. While such diagnostic approaches rely on high resolution imaging of the cell nucleus using expensive microscopy systems, developing portable mobile microscopes to visualize nuclear and chromatin condensation patterns is desirable at clinical settings with limited infrastructure. In this study, we develop a portable fluorescent mobile microscope capable of acquiring high resolution images of the nucleus and chromatin. Using this we extracted nuclear morphometric and chromatin texture based features and were able to discriminate between normal and cancer cells with similar accuracy as wide-field fluorescence microscopy. We were also able to detect subtle changes in nuclear and chromatin features in cells subjected to compressive forces, cytoskeletal perturbations and cytokine stimulation, thereby highlighting the sensitivity of the portable microscope. Taken together, we present a versatile platform to exploit nuclear morphometrics and chromatin condensation features as physical biomarkers for point-of-care diagnostic solutions.

Development and biological validation of a cyclic stretch culture system for the ex vivo engineering of tendons.

INTRODUCTION: An innovative approach to the treatment of tendon injury or degeneration is given by engineered grafts, made available through the development of bioreactors that generate tendon tissue in vitro, by replicating in vivo conditions. This work aims at the design of a bioreactor capable of applying a stimulation of cyclic strain on cell constructs to promote the production of bioartificial tissue with mechanical and biochemical properties resembling those of the native tissue. METHODS: The system was actuated by an electromagnet and design specifications were imposed as follows. The stimulation protocol provides to scaffolds a 3% preload, a 10% deformation, and a stimulation frequency rate set at 0.5, 1, and 2 Hz, which alternates stimulation/resting phases. Porcine tenocytes were seeded on collagen scaffolds and cultured in static or dynamic conditions for 7 and 14 days. RESULTS: The culture medium temperature did not exceed 37°C during prolonged culture experiments. The applied force oscillates between 1.5 and 4.5 N. The cyclic stimulation of the engineered constructs let both the cells and the scaffold fibers align along the strain direction in response to the mechanical stimulus. CONCLUSION: We designed a pulsatile strain bioreactor for tendon tissue engineering. The in vitro characterization shows a preferential cell alignment at short time points. Prolonged culture time, however, seems to influence negatively on the survival of the cells indicating the need of further optimization concerning the culture conditions and the mechanical stimulation.