Louise P. Cramer

An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism

BACKGROUND Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier? RESULTS We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded. CONCLUSIONS We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.

Myosin II-dependent cortical movement is required for centrosome separation and positioning during mitotic spindle assembly

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.

ADF/Cofilin Controls Cell Polarity during Fibroblast Migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.

Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide

BACKGROUND In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.

Actin at cell-cell junctions is composed of two dynamic and functional populations

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.

Actomyosin II contractility expels von Willebrand factor from Weibel–Palade bodies during exocytosis

High-resolution microscopy reveals how discrete actin cytoskeletal functions inhibit or promote specific exocytic steps during regulated secretion.

Forming the cell rear first: breaking cell symmetry to trigger directed cell migration

Directed cell migration requires the breaking of cell symmetry to generate a cell front and a cell rear along an axis approximately aligned with the direction of locomotion. In most cell types, regulated actin polymerization promotes initial cell front formation and its subsequent persistent protrusion, whereas myosin II-based forces are required to initially create and then maintain the cell rear. Molecular models for cell migration have focused extensively on cell protrusion, and the breaking of cell symmetry is almost universally portrayed with the cell front forming first. Although data supports this model for cells moving towards chemo-attractants, in the absence of any guidance cue, cell symmetry is broken by the cells constitutively forming the cell rear first. This allows an alternative model for triggering cell migration starting with retraction at the back of the cell. In this model, actomyosin II activity within the cell body and prospective cell rear occurs before a spatial bias in actin polymerization at the cell front. Creating the cell rear first may be a useful tool employed by a wide-range of migrating cell types, particularly when moving away from repellent cues.

Retrograde Flow and Myosin II Activity within the Leading Cell Edge Deliver F-Actin to the Lamella to Seed the Formation of Graded Polarity Actomyosin II Filament Bundles in Migrating Fibroblasts

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.

Actin coats and rings promote regulated exocytosis

It is well known that actin can associate with intracellular membranes to drive endocytosis and the rocketing motion of bacteria, virions and some organelles and to regulate synaptic vesicle plasticity. Actin also has been extensively reported to be involved at several steps of exocytosis; however, it has typically been described as functioning either within the actin cortex or by providing actin tracks for organelle transport. Increasingly, actin filament coats or rings have been directly localized on the surface of the exocytic organelle. Here, we suggest a common mechanism for actin-based regulation of large secretory granules whereby organelle-associated actomyosin II contraction either directly expels secretory content or stabilizes the exocytosing organelle.

ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization

How formation of the front and rear of a cell are coordinated during cell polarization in migrating cells is not well understood. Time-lapse microscopy of live primary chick embryo heart fibroblasts expressing GFP-actin show that, prior to cell polarization, polymerized actin in the cell body reorganizes to form oriented actin-filament bundles spanning the entire cell body. Within an average of 5 minutes of oriented actin bundles forming, localized cell-edge retraction initiates at either the side or at one end of the newly formed bundles and then elaborates around the nearest end of the bundles to form the cell rear, the first visual break in cell symmetry. Localized net protrusion occurs at the opposing end of the bundles to form the cell front and lags formation of the rear of the cell. Consequently, cells acquire full polarity and start to migrate in the direction of the long axis of the bundles, as previously documented for already migrating cells. When ADF/cofilin family protein activity or actin-filament disassembly is specifically blocked during cell polarization, reorganization of polymerized actin to form oriented actin-filament bundles in the cell body fails, and formation of the cell rear and front is inhibited. We conclude that formation of oriented actin-filament bundles in the cell body requires ADF/cofilin family proteins, and is an early event needed to coordinate the spatial location of the cell rear and front during fibroblast polarization.