Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics.

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.

Increased synapsin expression and neurite sprouting in lamprey brain after spinal cord injury.

Spinal cord injury induces structural plasticity throughout the mammalian nervous system, including distant locations in the brain. Several types of injury-induced plasticity have been identified, such as neurite sprouting, axon regeneration, and synaptic remodeling. However, the molecular mechanisms involved in injury-induced plasticity are unclear as is the extent to which injury-induced plasticity in brain is conserved across vertebrate lineages. Due to its robust roles in neurite outgrowth and synapse formation during developmental processes, we examined synapsin for its potential involvement in injury-induced plasticity. We used lamprey, a vertebrate that undergoes robust anatomical plasticity and functional recovery after spinal cord injury. At 3 and 11 weeks after spinal cord transection, synapsin I mRNA was upregulated >2-fold in lamprey brain, as assayed by semi-quantitative RT-PCR. Other synaptic vesicle-associated genes remained unchanged. In situ hybridization revealed that synapsin I mRNA was increased globally throughout the lamprey brain. Immunolabeling for synapsin I protein revealed a significant increase in both the intensity and density of synapsin I-positive structures in lamprey hindbrain at 11 weeks post-transection, relative to controls. Moreover, the number of structures immunolabeled for phospho-synapsin (serine 9) increased after injury, suggestive of neurite sprouting. Indeed, at the ultrastructural level, there was an increase in neurite density at 11 weeks post-transection. Taken together, these data show that neurite sprouting in the brain is an evolutionarily conserved response to a distant spinal cord injury and suggest that synapsin and its phosphorylation at serine 9 play key roles in the sprouting mechanism.

Diversity and evolution of coral fluorescent proteins.

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.