Multiple attributes of the three-dimensional (3D) extracellular matrix (ECM) have been independently implicated as regulators of cell motility, including pore size, crosslink density, structural organization, and stiffness. However, these parameters cannot be independently varied within a complex 3D ECM protein network. We present an integrated, quantitative study of these parameters across a broad range of complex matrix configurations using self-assembling 3D collagen and show how each parameter relates to the others and to cell motility. Increasing collagen density resulted in a decrease and then an increase in both pore size and fiber alignment, which both correlated significantly with cell motility but not bulk matrix stiffness within the range tested. However, using the crosslinking enzyme Transglutaminase II to alter microstructure independently of density revealed that motility is most significantly predicted by fiber alignment. Cellular protrusion rate, protrusion orientation, speed of migration, and invasion distance showed coupled biphasic responses to increasing collagen density not predicted by 2D models or by stiffness, but instead by fiber alignment. The requirement of matrix metalloproteinase (MMP) activity was also observed to depend on microstructure, and a threshold of MMP utility was identified. Our results suggest that fiber topography guides protrusions and thereby MMP activity and motility.

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that plays an important role in long bone development. The G380R mutation in FGFR3 transmembrane domain is known as the genetic cause for achondroplasia, the most common form of human dwarfism. Despite many studies, there is no consensus about the exact mechanism underlying the pathology. To gain further understanding into the physical basis behind the disorder, here we measure the activation of wild-type and mutant FGFR3 in mammalian cells using Western blots, and we analyze the activation within the frame of a physicalchemical model describing dimerization, ligand binding, and phosphorylation probabilities within the dimers. The data analysis presented here suggests that the mutation does not increase FGFR3 dimerization, as proposed previously. Instead, FGFR3 activity in achondroplasia is increased due to increased probability for phosphorylation of the unliganded mutant dimers. This finding has implications for the design of targeted molecular treatments for achondroplasia.Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase that consists of an extracellular domain with three immunoglobulin-like motifs, a single transmembrane (TM) 2 domain, and an intracellular split tyrosine kinase domain. Ligands (fibroblast growth factors (fgfs)) and heparin bind to the extracellular domains and stabilize the dimer, with the ligand inducing a conformational change in the extracellular domain (1). The contact stimulates catalytic activity and results in the cross-phosphorylation of the two receptors. This activates the catalytic domains for the phosphorylation of cytoplasmic substrates and triggers signaling cascades (2, 3).Mutations in FGFR3 are known to affect long bone development, which proceeds via endochondral ossification along a pathway involving differentiation of mesenchymal stem cells into cartilage, followed by bone invasion (as the chondrocytes of the cartilage die, the space is invaded by bone). FGFR3 works as a negative regulator of bone development by mediating prodifferentiation signals in chondrocytes (4 -6). FGFR3 overactivation because of mutations alters the terminal differentiation into hypertrophic chondrocytes, effectively shortening the proliferation phase. Furthermore, FGFR3 overactivation can cause cancers of the epithelium (7).The G380R mutation in FGFR3 TM domain has been linked to achondroplasia, the most common form of human dwarfism (8). The incidence rate of achondroplasia is about 1/15,000 live births (5). It is an autosomal dominant disorder that interferes with the maturation of the cartilage growth plate of long bones (6, 9). The phenotype is characterized by short stature, narrowing of the lumbar spinal canal, accentuated bowing of the middle and lower part of the back, and trident-shaped hands (5). Affected individuals often exhibit other skeletal as well as neurological complications.The physical basis underlying achondroplasia is still under debate, and multiple mechanisms may be contributi...

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Plasma membrane derived vesicles are used as a model system for the biochemical and biophysical investigations of membrane proteins and membrane organization. The most widely used vesiculation procedure relies on formaldehyde and dithiothreitol (DTT), but these active chemicals may introduce artifacts in the experimental results. Here we describe a procedure to vesiculate Chinese hamster ovary (CHO) cells, widely used for the expression of recombinant proteins, using a hypertonic vesiculation buffer containing chloride salts and no formaldehyde or DTT. We characterize the size distribution of the produced vesicles. We also show that these vesicles can be used for the biophysical characterization of interactions between membrane proteins.

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