Paul C. Bridgman

Capping protein levels influence actin assembly and cell motility in dictyostelium

Actin assembly is important for cell motility, but the mechanism of assembly and how it relates to motility in vivo is largely unknown. In vitro, actin assembly can be controlled by proteins, such as capping protein, that bind filament ends. To investigate the function of actin assembly in vivo, we altered the levels of capping protein in Dictyostelium cells and found changes in resting and chemoattractant-induced actin assembly that were consistent with the in vitro properties of capping protein in capping but not nucleation. Significantly, overexpressers moved faster and underexpressers moved slower than control cells. Mutants also exhibited changes in cytoskeleton architecture. These results provide insights into in vivo actin assembly and the role of the actin cytoskeleton in motility.

Structural abnormalities develop in the brain after ablation of the gene encoding nonmuscle myosin II-B heavy chain.

Ablation of nonmuscle myosin heavy chain II‐B (NMHC‐B) in mice results in severe hydrocephalus with enlargement of the lateral and third ventricles. All B‐/B‐ mice died either during embryonic development or on the day of birth (PO). Neurons cultured from superior cervical ganglia of B‐/B‐ mice between embryonic day (E) 18 and P0 showed decreased rates of neurite outgrowth, and their growth cones had a distinctive narrow morphology compared with those from normal mice. Serial sections of E12.5, E13.5, and E15 mouse brains identified developmental defects in the ventricular neuroepithelium. On E12.5, disruption of the coherent ventricular surface and disordered cell migration of neuroepithelial and differentiated cells were seen at various points in the ventricular walls. These abnormalities resulted in the formation of rosettes in various regions of the brain and spinal cord. On E13.5 and E15, disruption of the ventricular surface and aberrant protrusions of neural cells into the ventricles became more prominent. By E18.5 and P0, the defects in cells lining the ventricular wall resulted in an obstructive hydrocephalus due to stenosis or occlusion of the third ventricle and cerebral aqueduct. These defects may be caused by abnormalities in the cell adhesive properties of neuroepithelial cells and suggest that NMHC‐B is essential for both early and late developmental processes in the mammalian brain. J. Comp. Neurol. 433:62–74, 2001.

Laminin stimulates and guides axonal outgrowth via growth cone myosin II activity

Guidance cues and signal transduction mechanisms acting at the nerve growth cone are fairly well understood, but the intracellular mechanisms operating to change the direction of axon outgrowth remain unknown. We now show that growth cones integrate myosin II–dependent contraction for rapid, coordinated turning at borders of laminin stripes in response to signals from laminin-activated integrin receptors; in the absence of myosin II activity, outgrowth continues across the borders.

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.

Retrograde flow rate is increased in growth cones from myosin IIB knockout mice

Growth cones of myosin-IIB-knockout mice have reduced outgrowth rates and traction force. There is a close relationship between traction force, retrograde flow and forward advance of growth cones. All three activities appear to be at least partially myosin dependent. Therefore, we have now tested for differences in retrograde flow rates between growth cones from myosin-IIB-knockout mice and their normal littermates. By placing nerve-growth-factor-coated silica beads on the surface of growth cones with laser tweezers, or by tracking GFP-myosin IIA spots, we found that the retrograde flow rate was increased more than two fold in the knockout growth cones compared with the wild type. These data suggest that both myosin IIA and IIB normally contribute to retrograde flow and the properties of the flow are strongly influenced by myosin IIB because of its location and abundance. However, in the absence of myosin IIB, myosin IIA takes over this function. The change in retrograde flow rate may reflect the difference in functional properties of these two myosins. Knockout growth cones also exhibited reduced stability of lamellipodia, possibly as a partial consequence of this increased retrograde flow rate. In addition, microtubules penetrated a shorter distance into filopodia, which suggests that the increase in flow rate may adversely affect the microtubule-dependent maturation of filopodia. Taken together these data support the idea that the forward advance of the growth cone is myosin II dependent and involves multiple myosin II isoforms.

Neurite outgrowth on electrospun nanofibers with uniaxial alignment: The effects of fiber density, surface coating, and supporting substrate

Electrospun nanofibers with uniaxial alignment have recently gained its popularity as scaffolds for neural tissue engineering. Many studies have demonstrated that the nanofibers could guide the neurites to extend along the direction of alignment, resembling the native hierarchy of the nerve tissue. However, the contact cues provided by the nanofibers can be far more complicated than just guiding the neurites to extend along them. In the current study, we used dorsal root ganglia as a model system to systematically investigate the interactions between neurites and uniaxially aligned nanofibers. We demonstrated, for the first time, that the neurites could not only project along the nanofibers, but also be directed to grow along a direction perpendicular to the aligned nanofibers, depending on the following parameters: (i) the density of nanofibers, (ii) the protein deposited on the surfaces of the nanofibers, and (iii) surface properties of the substrate on which the nanofibers were supported. We also investigated the pharmacological effect of myosin II inhibition on the nanofiber-guided growth of neurites by adding blebbistatin to the culture medium. Our findings offer new insights into the design of nanofiber-based scaffolds for nerve injury repair and will provide new guidelines for the construction of well-defined neuronal network architecture (the so-called neural circuits).

Role of Myosin II in Axon Outgrowth

The initial stages of nerve outgrowth carried out by growth cones occur in three fundamental cyclic steps. Each of these steps appears to require myosin II activity to variable degrees. The steps include the following: (a) exploration, involving extensions and retractions that are driven and controlled by the interaction of actin retrograde flow and polymerization; (b) adhesion of new extensions to the substrate, which has been shown to be mediated by complex interactions between extracellular matrix proteins, cell adhesion proteins, and the actin cytoskeleton; and (c) traction force generated during forward advance of the growth cone, resulting in the production of tension on the neurite.

Mammalian myosin Iα is concentrated near the plasma membrane in nerve growth cones

To determine if unconventional myosins play a role in nerve outgrowth, antibodies specific for rat brain derived mammalian myosin I alpha (MMI alpha) were used to label cultured rat superior cervical ganglion nerve cells. Observations were made at both the light and electron microscopic level of resolution using preparative procedures designed to enhance the ability to precisely determine the relationship between antibody label and cellular structures in order to map the distribution and structural association of this myosin. Immunofluorescence showed that MMI alpha has a punctate distribution throughout the nerve cell body, neurites, and growth cones. In growth cones, MMI alpha staining is sometimes elevated in thin peripheral regions of high actin content at the leading edge. Immunoelectron microscopy using colloidal gold conjugated antibodies showed that in growth cones MMI alpha is absent from membranous organelles and is concentrated primarily in the cell cortex adjacent to the cell membrane. The cortical label is equally distributed between upper and lower membranes. The plasma membrane association of the MMI alpha label persists under conditions in which the actin cytoskeleton is perturbed or removed, suggesting a direct association between a fraction of MMI alpha and the plasma membrane. MMI alpha label is also associated with the non-cortical actin cytoskeleton. Partial disruption of the actin cytoskeleton using cytochalasin B causes redistribution of only a subset of MMI alpha label. These data suggest a complex relationship between MMI alpha, the actin cytoskeleton, and the plasma membrane in the growth cone. In contrast to its localization in the growth cone, in neuronal cell bodies MMI alpha is also associated with tubulovesicular structures. This suggests that at this location MMI alpha may either act as an organelle motor or is passively transported to the plasma membrane on vesicles.

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca(2+) dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

Myosin-II Negatively Regulates Minor Process Extension and the Temporal Development of Neuronal Polarity

The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated “minor processes” (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live‐cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA‐kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell‐permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis.