Elvis Pandzic

STICCS reveals matrix-dependent adhesion slipping and gripping in migrating cells.

Two-color spatio-temporal image cross-correlation spectroscopy (STICCS) is a new, to our knowledge, image analysis method that calculates space-time autocorrelation and cross-correlation functions from fluorescence intensity fluctuations. STICCS generates cellular flow and diffusion maps that reveal interactions and cotransport of two distinct molecular species labeled with different fluorophores. Here we use computer simulations to map the capabilities and limitations of STICCS for measurements in complex heterogeneous environments containing micro- and macrostructures. We then use STICCS to analyze the co-flux of adhesion components in migrating cells imaged using total internal reflection fluorescence microscopy. The data reveal a robust, time-dependent co-fluxing of certain integrins and paxillin in adhesions in protrusions when they pause, and in adhesions that are sliding and disassembling, demonstrating that the molecules in these adhesions move as a complex. In these regions, both α6β1- or αLβ2-integrins, expressed in CHO.B2 cells, co-flux with paxillin; an analogous cotransport was seen for α6β1-integrin and α-actinin in U2OS. This contrasts with the behavior of the α5β1-integrin and paxillin, which do not co-flux. Our results clearly show that integrins can move in complexes with adhesion proteins in protrusions that are retracting.

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function.

Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area.

Osmotically Challenging Single Escherichia Coli Cells: 1,2,3,Ready,Burst!

We use high spatial and temporal resolution spinning-disk confocal microscopy, fluorescent-labeling strategies, and automated image analysis to investigate the response of the mechanosensitive channel MscL to osmotic stress in living E. coli bacteria. We establish the viability of individual cells using a red-fluorescent nucleic acid stain, propidium iodide, and correlate it with cellular levels of EGFP-tagged MscL. We demonstrate that MscL promotes integrity of the cell membrane in the face of environmental osmotic pressure. For these experiments, a micro-fluidic device with temperature control and multi-generational capability was developed to determine which cells are viable and dividing following exposure to osmotic stress, and to study the ability of cells to recover from temporally varying stress stimuli.