Louise P. Cramer

The Randall Institute, Kings College London, 26-29 Drury Lane, London WC2B 5RL, UK.

Received 5/21/97; Accepted 5/26/97

2. INTRODUCTION

A number of different types of motility occur in eukaryotic cells. In motile, eukaryotic cells adhering to solid substrata, one of the most dramatic is the continuous flow of cell-associated material, directed inwards from the cell periphery, both over the cell surface and inside the cell. In the literature, this flow is variously termed inward, centripetal, backward, or retrograde, flux or flow. Different types of flow of cell-associated material in eukaryotic cells has been observed for about two centuries, and is a fundamental property of all eukaryotic cells so far studied. In recent years, retrograde flow in adherent, motile, eukaryotic cells has been intensely investigated, although its function, remains for the most part, unknown. In these cells, observed retrograde flow may occur either relative to the cell (Fig 1A) or relative to the substratum (Fig 1B).

Fig 1. Types of retrograde flow in adherent, motile, eukaryotic cells. (A, relative to the cell) Top panel: cell-associated material (black sphere) is essentially stationary relative to the substratum (fixed point, X), as the cell (oblong) physically moves forward (arrow). Middle panel: if only the front of the cell moves forward, the material appears to flow retrograde from the cell front. Bottom panel, if all of the cell moves forward (cell locomotion) the material appears to flow retrograde to the back of the cell. This occurs for certain cell surface receptors in locomoting cells (termed capping). (B, relative to the substratum) (focus of this review) Cell-associated material (top panel, black sphere) physically flows retrograde (to new position, bottom panel), relative to a fixed point (X) on the substratum. This can occur(bottom panel) on both stationary (e.g., solid oblong) and moving (e.g., solid and dashed oblong), motile cells.

An adherent, eukaryotic, motile cell is composed of several distinct cell regions (Fig 2). At the front of the cell are leading edge structures. These comprise the lamellipodium (a thin cellular band, typically less than 0.5 mm thick, and 1-10 mm long from front to back), and long, thin, cylindrical extensions of the lamellipodium termed filopodia, or microspikes which are shorter. Behind the lamellipodium is a thicker cell region termed the lamella. Behind the lamella is the cell body, which is the bulkiest cell region comprising the nucleus and most of the organelles. Retrograde flow can occur in all of these cell regions (e.g. Fig 2 illustrates retrograde flow relative to the substratum in lamellipodia and lamella). Also, retrograde flow is opposite to, and sometimes occurs simultaneously with, several types of forward cell motility (Fig 2). These are: protrusion which brings leading edge structures forward; cell body motility or traction which brings the bulk of the cell and nucleus forward; and tail retraction/ deadhesion which brings the rear of the cell forward.

Fig 2. Cell regions and types of motility in motile cells. (A, motile/locomoting cell) In a fibroblast the lamellipodium is often raised up off the substratum. In certain other motile cell types such as keratocytes the lamellipodium is in constant contact with the substratum (as drawn in B). Retrograde flow relative to the substratum has mostly been studied for individual types of cell-associated material crossing the lamellipodium (thick arrow to left). Retrograde flow occurs opposite to the direction of certain types of forward cell motility that may occur in motile cells (protrusion, cell body motility and tail retraction). Strictly a motile cell is termed a locomoting cell, only if it undergoes net, protrusion, cell body motility and tail retraction, such that the entire cell boundary moves to a new position (e.g. Fig 1A, compare oblong, top and bottom panels). (B, neuronal growth cone) A growth cone is similarly organized to a motile cell except the nucleus is not located in the growth cone body, and the neurite replaces the tail. In the literature, the lamellipodium and lamella are sometimes collectively referred to as 'peripheral domain' and the growth cone body as 'central domain'. Growth cone body motility is sometimes referred to as central domain extension. Similarly, to locomote, a motile growth cone must undergo, net, protrusion, growth cone body motility and deadhesion/neurite extension. In contrast to motile cells, in growth cones, retrograde flow relative to the substratum has been mostly studied for individual types of cell-associated material crossing both the lamellipodium (thick arrow to left) and lamella (dashed thick arrow to left), and recently, mostly in Aplysia bag cell neurons.

In the lamella and cell body the type of retrograde flow most understood typically occurs relative to the cell in locomoting cells. This is capping of cell surface receptors. Surface receptors flowing from the lamella and cell body cap over the nucleus or cell tail. From genetic studies, capping requires myosin II (1-3). Retrograde flow relative to the substratum in the lamella and cell body has in general been less studied. At least for certain types of cell-associated material, it is known that this is driven by a myosin (Waterman-Storer and Salmon, submitted), but not myosin II (3). In contrast in leading edge structures, mostly in lamellipodia, retrograde flow relative to the substratum, both over the lamellipodium surface and inside the lamellipodium, has been well studied. In both protruding and stationary lamellipodia, a variety of cell-associated material, including actin filaments, flows retrograde relative to the substratum (Fig 3). I will collectively refer to this material, except actin filaments, as particles. To distinguish between flow of particles and flow of actin filaments, I will use the terms 'retrograde particle flow', and 'retrograde actin flow', respectively. Many mechanisms have been proposed to drive retrograde particle flow in lamellipodia (4-7). It is now widely accepted that actin filaments are required to generate force to drive retrograde particle flow. Compelling evidence is that poisons of actin inhibit retrograde particle flow in lamellipodia (3, 8-10).

Early ideas on how actin filaments generated force to drive retrograde particle flow in lamellipodia were theoretical. One quite popular idea was that contraction of an actin filament network moved the lipid bilayer of a lamellipodium backward as a sheet, and structures on the moving sheet rode as passengers (4, 11). At the time this made sense; flow of particles on the surface of lamellipodia were thought to reflect a moving cell surface, and muscle proteins were just beginning to be identified in non-muscle motile cells (reviewed in (12)). This theory was not pursued once Singer and Nicholson (13) introduced the idea that the lipid bilayer was fluid. Of the several alternative explanations offered, the one that turned out to be the most pertinent came from a discussion between Wolpert and Harris in 1973 (11). Wolpert hypothesized that a 'filamentous system' directly moved particles retrograde. Precisely how has been debated since this time. Part of the problem is that over the last 10 years or so different types of particles have been studied in different motile cell types. For example, the tendency has been to view particles flowing retrograde on the cell surface, as the same phenomenon as particles and actin filaments flowing retrograde inside the lamellipodium. It may turn out, however, that retrograde flow of particular types of particles associated with lamellipodia in some motile cell types, may be a separate phenomenon, driven by a distinct mechanism. Perhaps related to this, different results have been obtained in different motile cell types, particularly in Aplysia bag cell neuronal growth cones, fibroblasts and keratocytes. This has led to distinct views on both the mechanism of retrograde particle flow, and function of retrograde actin flow in lamellipodia.

Fig 3. Different types of cell-associated material that flow retrograde relative to the substratum in lamellipodia. In motile and locomoting cells, retrograde flow (long arrow to left) is directed from the front to the back of both protruding (shorter arrow to right) and stationary lamellipodia, and other leading edge structures. Flowing retrograde over the surface of lamellipodia are: membrane ruffles, characteristic of fibroblasts due to lamellipodia that lift up off the substratum and flow retrograde; foreign-attached particles (e.g. beads, glass fragments); cell surface receptors; nodules; and blebs. These are shown flowing over the dorsal surface, but some, e.g. foreign-attached particles, have also been observed to flow over the ventral surface. Flowing retrograde inside the cell are: phase dense inhomogeneities; and fibrous material, including in most cell types studied, actin filaments.

In this review, I will briefly describe the organization of actin filaments in leading edge structures of adherent, motile cells, and in neuronal growth cones. Then, I will describe potential types of actin-dependent motile force to drive retrograde particle flow relative to the substratum in lamellipodia of these cells, and in growth cones of Aplysia neurons. I will present evidence in favor of each type of motile force, and discuss function of retrograde actin flow.