Zvi Kam

Dynamics and segregation of cell–matrix adhesions in cultured fibroblasts

Here we use time-lapse microscopy to analyse cell–matrix adhesions in cells expressing one of two different cytoskeletal proteins, paxillin or tensin, tagged with green fluorescent protein (GFP). Use of GFP–paxillin to analyse focal contacts and GFP–tensin to study fibrillar adhesions reveals that both types of major adhesion are highly dynamic. Small focal contacts often translocate, by extending centripetally and contracting peripherally, at a mean rate of 19 micrometres per hour. Fibrillar adhesions arise from the medial ends of stationary focal contacts, contain α5β1 integrin and tensin but not other focal-contact components, and associate with fibronectin fibrils. Fibrillar adhesions translocate centripetally at a mean rate of 18 micrometres per hour in an actomyosin-dependent manner. We propose a dynamic model for the regulation of cell–matrix adhesions and for transitions between focal contacts and fibrillar adhesions, with the ability of the matrix to deform functioning as a mechanical switch.

Dynamic Proteomics of Individual Cancer Cells in Response to a Drug

Why do seemingly identical cells respond differently to a drug? To address this, we studied the dynamics and variability of the protein response of human cancer cells to a chemotherapy drug, camptothecin. We present a dynamic-proteomics approach that measures the levels and locations of nearly 1000 different endogenously tagged proteins in individual living cells at high temporal resolution. All cells show rapid translocation of proteins specific to the drug mechanism, including the drug target (topoisomerase-1), and slower, wide-ranging temporal waves of protein degradation and accumulation. However, the cells differ in the behavior of a subset of proteins. We identify proteins whose dynamics differ widely between cells, in a way that corresponds to the outcomes—cell death or survival. This opens the way to understanding molecular responses to drugs in individual cells.

A paxillin tyrosine phosphorylation switch regulates the assembly and form of cell-matrix adhesions

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.

Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing

Cell elongation and polarization are basic morphogenetic responses to extracellular matrix adhesion. We demonstrate here that human cultured fibroblasts readily polarize when plated on rigid, but not on compliant, substrates. On rigid surfaces, large and uniformly oriented focal adhesions are formed, whereas cells plated on compliant substrates form numerous small and radially oriented adhesions. Live-cell monitoring showed that focal adhesion alignment precedes the overall elongation of the cell, indicating that focal adhesion orientation may direct cell polarization. siRNA-mediated knockdown of 85 human protein tyrosine kinases (PTKs) induced distinct alterations in the cell polarization response, as well as diverse changes in cell traction force generation and focal adhesion formation. Remarkably, changes in rigidity-dependent traction force development, or focal adhesion mechanosensing, were consistently accompanied by abnormalities in the cell polarization response. We propose that the different stages of cell polarization are regulated by multiple, PTK-dependent molecular checkpoints that jointly control cell contractility and focal-adhesion-mediated mechanosensing.

On the crystallization of proteins.

Abstract We report on theoretical and experimental work aimed at a systematic approach to the crystallization of proteins. Successful crystallization depends on the competition between the growth rates for compact three-dimensional structures and long-chain structures leading to an amorphous precipitate. Quasi-elastic light scattering was used to monitor the size and shape distribution of small aggregates in a model system (lysozyme) during the pre-nucleation stage. With the aid of a simple model, the line-width of the scattered light was used to predict whether crystals or an amorphous precipitate would result. Once visible crystals appeared, the lysozyme concentration near the crystal surface was monitored and the kinetic parameters for growth obtained. A peculiar self-limiting phenomenon causes crystals to stop growing after a certain size has been reached. When these terminal size crystals were cleaved, growth occurred at the surface until the original size was approximately restored.

p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry

In this study the direct involvement of cadherins in adhesion-mediated growth inhibition was investigated. It is shown here that overexpression of N-cadherin in CHO cells significantly suppresses their growth rate. Interaction of these cells and two additional fibroblastic lines with synthetic beads coated with N-cadherin ligands (recombinant N-cadherin ectodomain or specific antibodies) leads to growth arrest at the G1 phase of the cell cycle. The cadherin-reactive beads inhibit the entry into S phase and the reduction in the levels of cyclin-dependent kinase (cdk) inhibitors p21 and p27, following serum-stimulation of starved cells. In exponentially growing cells these beads induce G1 arrest accompanied by elevation in p27 only. We propose that cadherin-mediated signaling is involved in contact inhibition of growth by inducing cell cycle arrest at the G1 phase and elevation of p27 levels.

Engineering vessel-like networks within multicellular fibrin-based constructs

Sufficient vascularization in engineered tissues can be achieved through coordinated application of improved biomaterial systems with proper cell types. In this study, we employed 3D fibrin gels alone or in combination with the synthetic poly(l-lactic acid) (PLLA)/polylactic-glycolic acid (PLGA) sponges to support in-vitro construct vascularization and to enhance neovascularization upon implantation. Two multicellular assays were embedded in these constructs: (a) co-culture of endothelial (EC) and fibroblast cells, and (b) a tri-culture combination of ECs, fibroblasts and tissue specific skeletal myoblast cells. In-vitro vessel network formation was examined under advanced confocal microscopy in various time points from cell seeding. Vessel network maturity levels and morphology were found to be highly regulated by fibrinogen concentrations in-vitro. Combination of PLLA/PLGA sponges with fibrin matrices provided added mechanical strength and featured highly mature vessels-like networks. Implantation studies revealed that the implanted ECs developed into 3D interconnected vessel-like networks in-vivo. The PLLA/PLGA scaffold proved to be a key stimulator of neovascularization and perfusion of implanted grafts. Our findings demonstrate that complex biomaterial platform involving fibrin and PLLA/PLGA synthetic scaffold provide a way to enhancing vascularization in-vitro and in-vivo.

Fast live simultaneous multiwavelength four-dimensional optical microscopy.

Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading.

Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer

Microscopy-based fluorescence resonance energy transfer (FRET) provides an opportunity to monitor molecular processes in the natural environment in live cells. Here we studied molecular interactions and tyrosine phosphorylation of paxillin, Crk-associated substrate (CAS), and focal adhesion kinase (FAK) in focal adhesions. For that purpose, these focal adhesion phosphoproteins, fused to cyan or yellow fluorescent proteins (CFP or YFP) were expressed in cultured fibroblasts. To assess the dynamics of tyrosine phosphorylation we used YFP- or CFP-tagged SH2 domain of pp60src (dSH2), which specifically binds to phosphotyrosine residues. FRET measurements, combined with immunolabeling with phosphospecific antibodies revealed that FAK, CAS and paxillin are tyrosine phosphorylated in early matrix adhesions and that FAK is in FRET proximity to CAS and paxillin in focal complexes and focal adhesions. Data suggest that paxillin incorporation into nascent focal complexes precedes its tyrosine phosphorylation, which then gradually increases. In cells treated with Rho-kinase inhibitors or expressing constitutively active Rac, focal complexes showed similar levels of paxillin tyrosine phosphorylation as seen in mature focal adhesions. Dynamic FRET-based examination indicated that paxillin phosphorylation occurs in specific areas (hotspots) within focal adhesions, whereas FAK phosphorylation is broadly distributed.

Computational adaptive optics for live three-dimensional biological imaging

Light microscopy of thick biological samples, such as tissues, is often limited by aberrations caused by refractive index variations within the sample itself. This problem is particularly severe for live imaging, a field of great current excitement due to the development of inherently fluorescent proteins. We describe a method of removing such aberrations computationally by mapping the refractive index of the sample using differential interference contrast microscopy, modeling the aberrations by ray tracing through this index map, and using space-variant deconvolution to remove aberrations. This approach will open possibilities to study weakly labeled molecules in difficult-to-image live specimens.