Kurt I. Anderson

Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells

BACKGROUNDnIn the cortical region of motile cells, the actin network rapidly reorganizes as required for movement in various directions and for cell-to-substrate adhesion. The analysis of actin network dynamics requires the combination of high-resolution imaging with a specific fluorescent probe that highlights the filamentous actin structures in live cells.nnnRESULTSnCombining total internal reflection fluorescence (TIRF) microscopy with a method for labeling actin filaments, we analyze the dynamics of actin patterns in the highly motile cells of Dictyostelium. A rapidly restructured network of single or bundled actin filaments provides a scaffold for the assembly of differentiated actin complexes. Recruitment of the Arp2/3 complex characterizes stationary foci with a lifetime of 7-10 s and traveling waves. These structures are also formed in the absence of myosin-II. Arp2/3-actin assemblies similar to those driving the protrusion of a leading edge form freely at the inner face of the plasma membrane.nnnCONCLUSIONSnThe actin system of highly motile cells runs far from equilibrium and generates a multitude of patterns within a dynamic filamentous network. Traveling waves are the most complicated patterns based on recruitment of the Arp2/3 complex. They are governed by the propagated induction of actin polymerization. We hypothesize that the actin system autonomously generates primordia of specialized structures such as phagocytic cups or lamellipodia. These primordia would represent an activated state of the actin system and enable cells to respond within seconds to local stimuli by chemotaxis or phagocytic-cup formation.

Assembling an actin cytoskeleton for cell attachment and movement

2. The ¢broblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 2.1. Lamellipodia as ¢lament factories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 2.2. Ventral stress ¢bre assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 2.3. Arcs and dorsal stress ¢bres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275 2.4. Concave cell edges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

Polyene-lipids: a new tool to image lipids

Microscopy of lipids in living cells is currently hampered by a lack of adequate fluorescent tags. The most frequently used tags, NBD and BODIPY, strongly influence the properties of lipids, yielding analogs with quite different characteristics. Here, we introduce polyene-lipids containing five conjugated double bonds as a new type of lipid tag. Polyene-lipids exhibit a unique structural similarity to natural lipids, which results in minimal effects on the lipid properties. Analyzing membrane phase partitioning, an important biophysical and biological property of lipids, we demonstrated the superiority of polyene-lipids to both NBD- and BODIPY-tagged lipids. Cells readily take up various polyene-lipid precursors and generate the expected end products with no apparent disturbance by the tag. Applying two-photon excitation microscopy, we imaged the distribution of polyene-lipids in living mammalian cells. For the first time, ether lipids, important for the function of the brain, were successfully visualized.

N-WASP deficiency impairs EGF internalization and actin assembly at clathrin-coated pits

WASP and WAVE family proteins promote actin polymerization by stimulating Arp2/3-complex-dependent filament nucleation. Unlike WAVE proteins, which are known to drive the formation of protrusions such as lamellipodia and membrane ruffles, vertebrate cell functions of WASP or N-WASP are less well established. Recent work demonstrated that clathrin-coated pit invagination can coincide with assembly of actin filaments and with accumulation of N-WASP and Arp2/3 complex, but the relevance of their recruitment has remained poorly defined. We employed two-colour total internal reflection microscopy to study the recruitment and dynamics of various components of the actin polymerization machinery and the epidermal growth factor receptor signalling machinery during clathrin-coated pit internalization in control cells and cells genetically deficient for functional N-WASP. We found that clathrin-coated pit endocytosis coincides with the recruitment of N-WASP, Arp2/3 complex and associated proteins, but not of WAVE family members. Actin accumulation at clathrin-coated pits requires the Arp2/3 complex, since Arp2/3 complex sequestration in the cytosol abolished any detectable actin assembly. The absence of N-WASP caused a significant reduction in the frequencies of actin and Arp2/3 complex accumulations at sites of clathrin-coated pit invagination and vesicle departure. Although N-WASP was not essential for Arp2/3-complex-mediated actin assembly at these sites or for EGF receptor-mediated endocytosis, N-WASP deficiency caused a marked reduction of EGF internalization. We conclude that the assembly of WASP subfamily proteins and associated factors at sites of clathrin-coated pit invagination amplifies actin accumulations at these sites promoting efficient internalization of ligands via clathrin-mediated endocytosis.

Observing structure, function and assembly of single proteins by AFM

Single molecule experiments provide insight into the individuality of biological macromolecules, their unique function, reaction pathways, trajectories and molecular interactions. The exceptional signal-to-noise ratio of the atomic force microscope allows individual proteins to be imaged under physiologically relevant conditions at a lateral resolution of 0.5-1nm and a vertical resolution of 0.1-0.2nm. Recently, it has become possible to observe single molecule events using this technique. This capability is reviewed on various water-soluble and membrane proteins. Examples of the observation of function, variability, and assembly of single proteins are discussed. Statistical analysis is important to extend conclusions derived from single molecule experiments to protein species. Such approaches allow the classification of protein conformations and movements. Recent developments of probe microscopy techniques allow simultaneous measurement of multiple signals on individual macromolecules, and greatly extend the range of experiments possible for probing biological systems at the molecular level. Biologists exploring molecular mechanisms will benefit from a burgeoning of scanning probe microscopes and of their future combination with molecular biological experiments.

Visualising the actin cytoskeleton.

The actin cytoskeleton is a dynamic filamentous network whose formation and remodeling underlies the fundamental processes of cell motility and shape determination. To serve these roles, different compartments of the actin cytoskeleton engage in forming specific coupling sites between neighbouring cells and with the underlying matrix, which themselves serve signal transducing functions. In this review, we focus on methods used to visualise the actin cytoskeleton and its dynamics, embracing the use of proteins tagged with conventional fluorophores and green fluorescent protein. Included also is a comparison of cooled CCD technology, confocal and 2‐photon fluorescence microscopy of living and fixed cells, as well as a critique of current procedures for electron microscopy. Microsc. Res. Tech. 47:3–17, 1999.

The three-dimensional dynamics of actin waves, a model of cytoskeletal self-organization

Actin polymerization is typically initiated at specific sites in a cell by membrane-bound protein complexes, and the resulting structures are involved in specialized cellular functions, such as migration, particle uptake, or mitotic division. Here we analyze the potential of the actin system to self-organize into waves that propagate on the planar, substrate-attached membrane of a cell. We show that self-assembly involves the ordered recruitment of proteins from the cytoplasmic pool and relate the organization of actin waves to their capacity for applying force. Three proteins are shown to form distinct three-dimensional patterns in the actin waves. Myosin-IB is enriched at the wave front and close to the plasma membrane, the Arp2/3 complex is distributed throughout the waves, and coronin forms a sloping layer on top of them. CARMIL, a protein that links myosin-IB to the Arp2/3 complex, is also recruited to the waves. Wave formation does not depend on signals transmitted by heterotrimeric G-proteins, nor does their propagation require SCAR, a regulator upstream of the Arp2/3 complex. Propagation of the waves is based on an actin treadmilling mechanism, indicating a program that couples actin assembly to disassembly in a three-dimensional pattern. When waves impinge on the cell perimeter, they push the edge forward; when they reverse direction, the cell border is paralyzed. These data show that force-generating, highly organized supramolecular networks are autonomously formed in live cells from molecular motors and proteins controlling actin polymerization and depolymerization.

Recent advances using green and red fluorescent protein variants

Fluorescent proteins have proven to be excellent tools for live-cell imaging. In addition to green fluorescent protein (GFP) and its variants, recent progress has led to the development of monomeric red fluorescent proteins (mRFPs) that show improved properties with respect to maturation, brightness, and the monomeric state. This review considers green and red spectral variants, their paired use for live-cell imaging in vivo, in vitro, and in fluorescence resonance energy transfer (FRET) studies, in addition to other recent “two-color” advances including photoswitching and bimolecular fluorescence complementation (BiFC). It will be seen that green and red fluorescent proteins now exist with nearly ideal properties for dual-color microscopy and FRET.

Actin and the coordination of protrusion, attachment and retraction in cell crawling

To crawl over a substrate a cell must first protrude in front, establish new attachments to the substrate and then retract its rear. Protrusion and retraction utilise different subcompartments of the actin cytoskeleton and operate by different mechanisms, one involving actin polymerization and the other myosin-based contraction. Using as examples the rapidly locomoting keratocyte and the slowly moving fibroblast we illustrate how over expression of one or the other actin subcompartments leads to the observed differences in motility. We also propose, that despite these differences there is a common coordination mechanism underlying the genesis of the actin cytoskeleton that involves the nucleation of actin filaments at the protruding cell front, in the lamellipodium, and the relocation of these filaments, via polymerization and flow, to the more posterior actin filament compartments.

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres

Calponin (4.1–5.9 μM, pig stomach) inhibited maximal shortening velocity (V max) by 20–25% with only minor influence on force in skinned smooth muscle from guinea‐pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N‐terminal 1–228 amino acids engineered using a mouse cDNA construct (5.4 μM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin‐myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross‐bridge time spent in the attached state.