Regulation of a formin complex by the microtubule plus end protein tea1p

© 2004 Feierbach et al. This article is distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License. The definitive version was published in Journal of Cell Biology 165 (2004): 697-707, doi:10.1083/jcb.200403090.The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end–binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3{Delta}bud6{Delta}tea1{Delta} triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.This research was supported by National Institutes of Health grant GM R01-GM56836, a research project grant from the American Cancer Society, a Nikon summer fellowship at the Marine Biological Laboratory (Woods Hole, MA) to F. Chang, and a National Institutes of Health postdoctoral fellowship (GM20283) to B. Feierbach

Alpha-Herpesvirus Infection Induces the Formation of Nuclear Actin Filaments

Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process

GFP-VP26 Co-Localizes with Nuclear Actin Filaments

<div><p>(A) Neurons were infected with PRV expressing GFP-VP26 and were fixed at time points shown. A single focal plane through the nucleus is shown, which can result in actin filaments appearing “discontinous” due to filaments weaving in and out of the plane of focus. An enlarged image of the nucleus (inset) is shown for clarity. Merged image was created in ImageJ and color adjusted linearly to appear yellow. Scale bar = 10 μm.</p><p>(B) Histogram shows percentage of cells with co-localized GFP-VP26 and nuclear actin filaments in cells positive for nuclear actin at time points indicated. This histogram represents the percentage of cells within a population that show co-localization (this histogram does not show the degree to which GFP-VP26 and nuclear actin filaments are co-localized within a given cell).</p></div