A high-resolution multimode digital microscope system.

This chapter describes the development of a high-resolution, multimode digital imaging system based on a wide-field epifluorescent and transmitted light microscope, and a cooled charge-coupled device (CCD) camera. The three main parts of this imaging system are Nikon FXA microscope, Hamamatsu C4880 cooled CCD camera, and MetaMorph digital imaging system. This chapter presents various design criteria for the instrument and describes the major features of the microscope components-the cooled CCD camera and the MetaMorph digital imaging system.

mDia2 regulates actin and focal adhesion dynamics and organization in the lamella for efficient epithelial cell migration.

Cell migration requires spatial and temporal regulation of filamentous actin (F-actin) dynamics. This regulation is achieved by distinct actin-associated proteins, which mediate polymerization, depolymerization, severing, contraction, bundling or engagement to the membrane. Mammalian Diaphanous-related (mDia) formins, which nucleate, processively elongate, and in some cases bundle actin filaments, have been extensively studied in vitro, but their function in the cell has been less well characterized.

SNX9 couples actin assembly to phosphoinositide signals and is required for membrane remodeling during endocytosis.

Multiple modes of endocytosis require actin-dependent remodeling of the plasma membrane; however, neither the factors linking these processes nor their mechanisms of action are understood. The sorting nexin, SNX9, localizes to clathrin-coated pits where it interacts with dynamin and functions in clathrin-mediated endocytosis. Here, we demonstrate that SNX9 also localizes to actin-rich structures implicated in fluid-phase uptake, including tubular membranes containing GPI-anchored proteins and dorsal membrane ruffles.

Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases.

Development, angiogenesis, wound healing, and metastasis all involve the movement of cells in response to changes in the extracellular environment. To determine whether caveolin-1 plays a role in cell migration, we have used fibroblasts from knockout mice. Caveolin-1-deficient cells lose normal cell polarity, exhibit impaired wound healing, and have decreased Rho and increased Rac and Cdc42 GTPase activities.

Differential transmission of actin motion within focal adhesions.

Cell migration requires the transmission of motion generated in the actin cytoskeleton to the extracellular environment through a complex assembly of proteins in focal adhesions. We developed correlational fluorescent speckle microscopy to measure the coupling of focal-adhesion proteins to actin filaments. Different classes of focal-adhesion structural and regulatory molecules exhibited varying degrees of correlated motions with actin filaments, indicating hierarchical transmission of actin motion through focal adhesions.

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability.

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules.

Decreased polarity and increased random motility in PtK1 epithelial cells correlate with inhibition of endosomal recycling.

Locomoting cells exhibit a constant retrograde flow of plasma membrane proteins from the leading edge towards the cell center, which, when coupled to substrate adhesion, may drive forward cell movement. Here, we aimed to test the hypothesis that, in epithelial cells, these plasma membrane components are delivered via a polarized endo/exocytotic cycle, and that their correct recycling is required for normal migration. To this end, we expressed in PtK1 cells cDNA constructs encoding GDP-restricted (S25N) and GTP-restricted (Q70L) mutants of Rab11b, a small GTPase that has been implicated in the late stage of recycling, where membrane components from the endosomal recycling compartment are transported back to the plasma membrane.

Spatiotemporal feedback between actomyosin and focal-adhesion systems optimizes rapid cell migration.

Cells exhibit a biphasic migration-velocity response to increasing adhesion strength, with fast migration occurring at intermediate extracellular matrix (ECM) concentration and slow migration occurring at low and high ECM concentration. A simple mechanical model has been proposed to explain this observation, in which too little adhesion does not provide sufficient traction whereas too much adhesion renders cells immobile. Here we characterize a phenotype for rapid cell migration, which in contrast to the previous model reveals a complex interdependence of subcellular systems that mediates optimal cell migration in response to increasing adhesion strength.

Quantitative fluorescent speckle microscopy of cytoskeleton dynamics.

Fluorescent speckle microscopy (FSM) is a technology used to analyze the dynamics of macromolecular assemblies in vivo and in vitro. Speckle formation by random association of fluorophores with a macromolecular structure was originally discovered for microtubules. Since then FSM has been expanded to study other cytoskeleton and cytoskeleton-binding proteins.

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1.

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs.

Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions.

Spatial regulation of CLASP affinity for microtubules by Rac1 and GSK3beta in migrating epithelial cells.

Proteins that in cells specifically bind to growing microtubule plus ends (+TIPs) are thought to play important roles in polarization of the cytoskeleton. However, most +TIPs do not show a bias of their microtubule-binding behavior toward different subcellular regions. Here, we examine the dynamics of the +TIP CLASP in migrating PtK1 epithelial cells.

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM).

The effects of collapsing factors on F-actin content and microtubule distribution of Helisoma growth cones.

Growth cone collapsing factors induce growth cone collapse or repulsive growth cone turning by interacting with membrane receptors that induce alterations in the growth cone cytoskeleton. A common change induced by collapsing factors in the cytoskeleton of the peripheral domain, the thin lamellopodial area of growth cones, is a decline in the number of radially aligned F-actin bundles that form the core of filopodia. The present study examined whether ML-7, a myosin light chain kinase inhibitor, serotonin, a neurotransmitter and TPA, an activator of protein kinase C, which induce growth cone collapse of Helisoma growth cones, depolymerized or debundled F-actin.

A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis.

Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy.

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion.

Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility.

Background: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.

Results: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy.

Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1.

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays.

Modulation of Rac localization and function by dynamin.

The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin(-2) function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated.

Conserved microtubule-actin interactions in cell movement and morphogenesis.

Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions--regulatory and structural.

Regulation of leading edge microtubule and actin dynamics downstream of Rac1.

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1.

A high-speed multispectral spinning-disk confocal microscope system for fluorescent speckle microscopy of living cells.

Fluorescent speckle microscopy (FSM) uses a small fraction of fluorescently labeled subunits to give macromolecular assemblies such as the cytoskeleton fluorescence image properties that allow quantitative analysis of movement and subunit turnover. We describe a multispectral microscope system to analyze the dynamics of multiple cellular structures labeled with spectrally distinct fluorophores relative to one another over time in living cells. This required a high-resolution, highly sensitive, low-noise, and stable imaging system to visualize the small number of fluorophores making up each fluorescent speckle, a means by which to switch between excitation wavelengths rapidly, and a computer-based system to integrate image acquisition and illumination functions and to allow a convenient interface for viewing multispectral time-lapse data.

Converging populations of f-actin promote breakage of associated microtubules to spatially regulate microtubule turnover in migrating cells.

Background: In migrating cells, the retrograde flow of filamentous actin (f-actin) from the leading edge toward the cell body is accompanied by the synchronous motion of microtubules (MTs, ), whose plus ends undergo net growth. Thus, MTs must depolymerize elsewhere in the cell to maintain polymer mass over time. The source and location of depolymerized MTs is unknown.