Nanoimaging of Focal Adhesion Dynamics in 3D

Abstract

Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the N&B analysis shows paxillin aggregated predominantly at these focal contacts which are next to collagen fibers. At those sites, actin showed slower apparent diffusion rate, which indicated that actin is either polymerizing or binding to the scaffolds in these locals. Our findings demonstrate that by multiplexing these techniques we have the ability to spatially and temporally quantify focal adhesion assembly and disassembly in 3D space and allow the understanding tumor cell invasion in a more complex relevant environment.

Mechanosensing via cell-matrix adhesions in 3D microenvironments

“…These findings suggest that the two-spring model of 2D mechanosensing theorized by Bell and Schwarz et al may exist in 3D 68, 69 . Chiu et al 70 revealed that both actin and paxillin demonstrate different diffusion rates inside and outside of adhesion sites in 3D collagen consistent with a clutch-like mechanism. For vinculin, however, the half-life FRAP recovery rate in porcine dermis is similar to 2D rates 70, 71 , suggesting protein-to-protein differences in regulation and response to different types of ECM.…”

“…Chiu et al 70 revealed that both actin and paxillin demonstrate different diffusion rates inside and outside of adhesion sites in 3D collagen consistent with a clutch-like mechanism. For vinculin, however, the half-life FRAP recovery rate in porcine dermis is similar to 2D rates 70, 71 , suggesting protein-to-protein differences in regulation and response to different types of ECM. Clearly, how adhesion dynamics respond to differences in ECMs remains to be fully characterized.…”

Emerging Trends in Information‐Driven Engineering of Complex Biological Systems

“…[128] However, nanoscale topography under physiological conditions in 3D is very difficult to assess in situ considering the hydrated state of many biomaterials. [130] Advances in fluorescent microscopy will help to bolster our understanding of how cells interact with material topology in 3D. Future advancements in environmental scanning electron microscopy (ESEM) or cryo-SEM techniques may address some of these shortcomings, and recent advancements in preserving aqueous, bio-based surfaces for imaging in ultrahigh vacuum can be employed to better assess the aqueous phase topology of biomaterials.…”

“…[129] Additionally, advancements in ultrahigh resolution fluorescent imaging technologies are helping to shed light on focal adhesion dynamics in 3D systems. [130] Advances in fluorescent microscopy will help to bolster our understanding of how cells interact with material topology in 3D.…”

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy

“…Tight confinement further distorts the distribution, because this boundary condition excludes the largest step sizes. The artifacts introduced by confinement and in-frame motion were not encountered in previous applications of image correlation spectroscopy, because those experiments focused on slow motion (26,(31)(32)(33)(34), or else minimized pixel dwell time, as in raster image correlation spectroscopy (29). On the other hand, here in the regime of fast, confined motion, the two independent effects of blur and confinement manifest themselves as two independent diffusion-coefficient measurement biases.…”