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Since expression of GFP in mammalian cells does not signicantly affect proliferation rate or other important cellular functions, it serves as an endogenous uorescent marker for spatial distribution or biochemistry.GFP fused to cytoskeletal proteins has provided new insights into the real time dynamics of cell structure.Thus, a composite material description more closely describes mechanical properties of the cytoplasm than one composed of a single cytoskeletal protein.Mice lacking vimentin exhibit impaired wound healing and owinduced arterial remodeling that may be related to defects in contractility and mechanical integrity at the cellular level.A D point spread function was measured experimentally, and a constrained iterative deconvolution algorithm was applied to arrays of optical sections.Resolution in restored images during ow chamber experiments was typically, nm in the xy FIGURE. Red uorescent microspheres attached to the coverslip under the cell monolayers and used as ducial reference markers were Naftifine hydrochloride visualized using a rhodamine lter set. Dual wavelength D image stacks were acquired every sfor min.A step change in ow was imposed so that wall shear stress imposed on the endothelium was dyn cm, and image acquisition continued at the same rate for an additional min.Stationary red uorescent microspheres were chosen as ducial markers of coverslip position, and the D positions of GFP uorescence volumes was normalized to those of the microspheres.These methods allowed quantitative D analysis of IF distribution during changes in uid shear stress forces, as detailed later.Highmagnication view of IF displacement during consecutive min intervals. In some regions, however, groups of laments were translated as a rigid body without mesh deformation.IF displacement Mepivacaine hydrochloride occurred in D at various heights above the coverslip.These observations demonstrated that onset of shear stress induced heterogeneous displacement and deformation of the IF network.Analysis of timelapse movies allowed direct measurement of displacement using endogenous morphological features.Tracking of the D positions of connections among IF segments as a function of time clearly demonstrated that ow onset induced a range of displacement patterns.Before ow onset, most network features displayed random uctuations around a constant average position.However, some segment connections were translated by as much as mm within min of ow onset and then maintained their new average position.Other connection points exhibited slower directional motion with approximately constant velocity.Since the patterns of displacement induced by ow onset varied, it is likely that the distribution of cytoskeletal deformation depended to some extent on the local network morphology and therefore reected local cytoplasmic mechanical properties.The D uorescence distribution functionf represented IF positions in space and time.The degree of overlap of uorescence intensity between two consecutive time points represents the average magnitude of IF displacement within the spatial region of interest.The displacement index DI was computed based on the spatial product moment cross correlation between two timesti andtj. The computed values of DI were mapped spatially and with respect to time, revealing both spatial and temporal patterns of owinduced IF displacement.During noow intervals, small values of DI were computed in most spatial regions of cells.Larger IF displacement may be expected near the luminal surface where shear forces act directly, and smaller displacement below the nucleus may represent relative structural stability in this area of the cell.

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