Matthew Raab

Crawling from soft to stiff matrix polarizes the cytoskeleton and phosphoregulates myosin-II heavy chain

Cytoskeletal polarization occurs in response to mechanosensing of a transition from soft to stiff matrix during migration and promotes dephosphorylation of myosin-IIA, rearward localization of myosin-IIB, and durotaxis.

Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via nuclear exit of a mechanorepressor

Scarring is a long-lasting problem in higher animals, and reductionist approaches could aid in developing treatments. Here, we show that co-polymerization of collagen-I with polyacrylamide produces minimal matrix models of scars (MMMS), in which fractal-fiber bundles segregate heterogeneously to the hydrogel subsurface. Matrix stiffens locally – as in scars – while allowing separate control over adhesive-ligand density. The MMMS elicits scar-like phenotypes from mesenchymal stem cells (MSCs): cells spread and polarize quickly, increasing nucleoskeletal lamin-A yet expressing the ‘scar marker’, smooth muscle actin (SMA) more slowly. Surprisingly, expression responses to MMMS exhibit less cell-to-cell noise than homogeneously stiff gels. Such differences from bulk-average responses arise because a strong SMA repressor, NKX2.5, slowly exits the nucleus on rigid matrices. NKX2.5 overexpression overrides rigid phenotypes, inhibiting SMA and cell spreading, while cytoplasm-localized NKX2.5 mutants degrade in well-spread cells. MSCs thus form a ‘mechanical memory’ of rigidity by progressively suppressing NKX2.5, thereby elevating SMA in a scar-like state.

Matrix elasticity in vitro controls muscle stem cell fate in vivo

Almost every laboratory that grows mammalian cells today grows their cells on tissue culture plastic, which was introduced to cell culture decades ago based on properties such as inertness, transparency, and so forth. However, plastic is rigid and unlike the many soft tissues in the body. Polymer gel systems that mimic the softness of various tissues have been developed over the past decade to test and understand the effects of rigidity on cells such as muscle cells. One recent study even shows that muscle stem cells expand much better in vitro on muscle-mimetic gels and that such cells prove optimal for engraftment in muscle.

Persistence-driven durotaxis:generic, directed motility in rigidity gradients

Cells move differently on substrates with different rigidities: the persistence time of their motion is higher on stiffer substrates. We show that this behavior-in and of itself-results in a net flux of cells directed up a soft-to-stiff gradient. Using simple random walk models with varying persistence and stochastic simulations, we characterize the propensity to move in terms of the durotactic index also measured in experiments. A one-dimensional model captures the essential features and highlights the competition between diffusive spreading and linear, wavelike propagation. Persistence-driven durokinesis is generic and may be of use in the design of instructive environments for cells and other motile, mechanosensitive objects.

Matrix rigidity regulates microtubule network polarization in migration

The microtubule organizing center (MTOC) frequently polarizes to a position in front of the nucleus during cell migration, but recent work has shown conflicting evidence for MTOC location in migratory polarized cells. Here, we show that subcellular localization of the MTOC is modulated by extracellular matrix stiffness. In scratch wound assays as well as single cell migration of mesenchymal stem cells (MSCs) the MTOC appears randomly positioned when cells are migrating on soft matrix, whereas on stiff matrix the MTOC is in front of the nucleus. The bulk of the microtubule density is also equally likely to be in front of or behind the nucleus on soft matrix, but it is polarized in front of the nucleus on stiff matrix. This occurred during cell migration with cells in interphase. During cytokinesis, the centrosomes polarize on either side of the chromosomes even on soft matrix, with MIIB localized strongly in the cleavage furrow which depolarizes only on soft matrix as cells exit cytokinesis. When cells are immobilized on micro‐patterns printed on the top of substrates of different stiffness, MIIB polarized if the matrix was sufficiently stiff similar to results with migrating cells. However, the MTOC was randomly positioned with respect to the nucleus independent of matrix stiffness. We deduce that cell migration is necessary to orient the MTOC in front of the nucleus and that matrix stiffness helps to drive cell polarization during migration.

Matrices engineered with gradients in rigidity guide stem cell migration and polarize the cytoskeleton

Tissues all have characteristic stiffness that can be represented with elastic modulus. The role this may have in guiding cell migration and in particular stem cell migration to areas of fibrotic matrix is not elucidated. Matrix with gradient in rigidity was used to guide stem cell migration as well as polarize the cells cytoskeleton.

Cells CRAWL from Soft to Stiff Surfaces as Myosin-II Polarizes the Cytoskeleton

Scarring in the heart after a myocardial infarction, or scarring in the skin after wounding - lead to rigidification of tissue through extensive collagen crosslinking and can also lead to homing of adherent mesenchymal stem cells (MSCs). ‘Durotaxis’ describes the tendency for a cell to crawl from a soft, collagen-coated gel to an adjacent stiff matrix, but clear evidence for accumulation of any cell type has been lacking as has insight into molecular mechanisms. We cultured human MSCs on matrices with scar-like gradients in elastic modulus (stiffness) that are on order of 10 Pa/micron and document a bias in migration toward the stiff matrix with proliferation-independent accumulation taking just a couple of days. As found with other cell types, MSCs on stiff substrates show myosin-IIB is polarized toward the rear while the centrosome and microtubule (MT)-network are polarized toward the front, but such polarization is surprisingly absent from cells on soft substrates. With myosin-II inhibition, we find cells on stiff matrix crawl faster whereas cells on soft matrix are initially impeded but then transition to motile cells as their centrosomes and MT-networks polarize as they would on stiff matrix. While myosin-II is required for contractility but not migration, MTs are required for any migration - including durotaxis - but contractility on gels remain intact after destabilization of MTs. The model gel results thus show that the progressive polarization of myosin-II on stiff substrates is particularly key to durotaxis. The broader relevance of this conclusion is tested with decellularized heart tissue that permits an examination of MSC adhesion, contraction, and migration. Decellularized heart tissue was stiffened with chemical crosslinking to increase elastic modulus measured with atomic force microscopy. We see that this increased stiffness alters cell migration behavior.