Catherine G. Galbraith

The relationship between force and focal complex development

To adhere and migrate, cells must be capable of applying cytoskeletal force to the extracellular matrix (ECM) through integrin receptors. However, it is unclear if connections between integrins and the ECM are immediately capable of transducing cytoskeletal contraction into migration force, or whether engagement of force transmission requires maturation of the adhesion. Here, we show that initial integrin–ECM adhesions become capable of exerting migration force with the recruitment of vinculin, a marker for focal complexes, which are precursors of focal adhesions. We are able to induce the development of focal complexes by the application of mechanical force to fibronectin receptors from inside or outside the cell, and we are able to extend focal complex formation to vitronectin receptors by the removal of c-Src. These results indicate that cells use mechanical force as a signal to strengthen initial integrin–ECM adhesions into focal complexes and regulate the amount of migration force applied to individual adhesions at localized regions of the advancing lamella.

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

We demonstrate live-cell super-resolution imaging using photoactivated localization microscopy (PALM). The use of photon-tolerant cell lines in combination with the high resolution and molecular sensitivity of PALM permitted us to investigate the nanoscale dynamics within individual adhesion complexes (ACs) in living cells under physiological conditions for as long as 25 min, with half of the time spent collecting the PALM images at spatial resolutions down to ∼60 nm and frame rates as short as 25 s. We visualized the formation of ACs and measured the fractional gain and loss of individual paxillin molecules as each AC evolved. By allowing observation of a wide variety of nanoscale dynamics, live-cell PALM provides insights into molecular assembly during the initiation, maturation and dissolution of cellular processes.

Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination

A key challenge when imaging living cells is how to noninvasively extract the most spatiotemporal information possible. Unlike popular wide-field and confocal methods, plane-illumination microscopy limits excitation to the information-rich vicinity of the focal plane, providing effective optical sectioning and high speed while minimizing out-of-focus background and premature photobleaching. Here we used scanned Bessel beams in conjunction with structured illumination and/or two-photon excitation to create thinner light sheets (<0.5 μm) better suited to three-dimensional (3D) subcellular imaging. As demonstrated by imaging the dynamics of mitochondria, filopodia, membrane ruffles, intracellular vesicles and mitotic chromosomes in live cells, the microscope currently offers 3D isotropic resolution down to ∼0.3 μm, speeds up to nearly 200 image planes per second and the ability to noninvasively acquire hundreds of 3D data volumes from single living cells encompassing tens of thousands of image frames.

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure.

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics (≈200 nm). Here, we report the use of two-color photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PA-FPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of ≈20–30 nm at acquisition times of 5–30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

Cell migration: regulation of force on extracellular-matrix-integrin complexes

Cell migration relies upon forces generated by the cell. Recent studies have provided new insights into the processes by which cells generate and regulate the forces applied to extracellular matrix (ECM)-bound integrins and have led us to the working model described here. In this model, ECM binding to integrins in the front of lamellipodia causes those integrins to attach to the rearward-moving cytoskeleton. Integrin-cytoskeleton attachments in the front are strengthened as a result of ECM rigidity, enabling the cell to pull itself forward. The reduction in contact area at the rear compared with that at the lamellipodium concentrates the traction forces in the rear on fewer integrin-ECM bonds, facilitating release. In such a model, cell pathfinding and motility can be influenced by ECM rigidity.

Forces on adhesive contacts affect cell function

Cellular forces acting on the adhesive contacts made with the extracellular matrix (ECM) contribute significantly to cell shape, viability, signal transduction and motility. In the past two years, research has determined how cell spreading influences cell viability as well as cytoskeletal organization. The cytoskeleton generates a level of tension against the ECM that is proportional to ECM stiffness. The strength of this tension exerted against the ECM affects the migratory speed of the cell.

Super-resolution microscopy at a glance

Advances in microscopy and cell biology are intimately intertwined, with new visualization possibilities often leading to dramatic leaps in our understanding of how cells function. The recent unprecedented technical innovation of super-resolution microscopy has changed the limits of optical

Cell-matrix adhesions on poly(vinyl alcohol) hydrogels.

Cell-matrix adhesions regulate cell morphology, intracellular signaling, gene expression, and phenotype. Understanding how different methods of attaching matrix proteins to substrates affect the molecular arrangement of these adhesions offers the possibility of controlling cell function and architecture. The goal of this study was to visualize and quantify the cell-matrix adhesions formed by human fibroblasts on the matrix protein fibronectin covalently attached to poly(vinyl) alcohol (PVA) hydrogels. These adhesions were then compared with the cell adhesions formed in routine cell culture on fibronectin noncovalently coated onto glass coverslips or those formed on fibronectin covalently immobilized onto glass coverslips. Cell adhesions were characterized by immunofluorescence confocal microscopy utilizing paxillin as a marker for focal adhesions and alpha(5) integrin as a marker for fibrillar adhesions. As expected, distinct focal and fibrillar adhesions were observed in routine cell culture on coverslips coated noncovalently with fibronectin. Cells cultured on fibronectin covalently linked to PVA demonstrated diminished spatial separation of paxillin and alpha(5) integrin, accompanied by a reduction in fibrillar adhesions and fibronectin fibrillogenesis. Cells on fibronectin covalently immobilized on glass displayed the strongest marker colocalization and the most complete loss of fibrillar adhesions and lack of fibrillogenesis. These results indicate that fibronectin-conjugated PVA promotes the formation of cell adhesion structures intermediate in composition between those formed on noncovalently attached and covalently immobilized fibronectin. Furthermore, they imply that bioactive polymers can selectively induce specific cell-matrix adhesions, a characteristic that may have consequences in various tissue-engineering applications.

Changes in single-molecule integrin dynamics linked to local cellular behavior

Single-molecule microscopy has the potential to link repeatable discrete molecular behaviors to changes in cellular behavior. The approach used here finds characteristic changes in integrin density and mobility that are linked to local cellular protrusion. Mutants show that the density changes can be separated from mobility changes.