Evidence of LIMK1, PKD1 and BARS involvement in Golgi outpost formation on Hippocampal neurons

JAUSORO I, QUASSOLLO G, WOJNACKI J, GASTALDI L, MARZOLO M.P, CONDE C, CACERES A.

Buenos Aires

Congreso; • 2012 ISN Special Conference “Synapses and dendritic spines in Elath and disease” International Society for Neurochemistry ISN; 2012

ISN International Society of Neuroscience & UCA Universidad Catolica

In most mammalian cells the Endoplasmic reticulum (ER) is distributed throughout the cytoplasm, whereas the Golgi network is localized in the perinuclear region close to the MTOC. In neurons, this arrangement is quite different, as the Golgi not only consists of the perinuclear membrane array, but also of discrete structures dispersed along dendrites termed Golgi outposts. The involvement of dendritic Golgi outposts in post-ER trafficking has been established. More importantly, it has recently been shown that NMDAR are sorted at the ER from AMPAR, bypassing the somatic Golgi and merging instead from outposts, suggesting that may serve as secretory platforms for local delivery of synaptic receptors, and therefore have a role in synaptic plasticity. Golgi outposts are also important for dendritic development. They are observed in fast growing dendrites both in vivo and in vitro and localized at branch points. Disruption of the dynein/dynactin adaptor complex or dynein mutations in Drosophila sensory neurons reduced the number of Golgi outposts and dendritic branches, demonstrating a direct relationship between their abundance and dendritic complexity. However, the crucial question of how the outposts are generated remains unanswered. Two possible scenarios have been proposed based on examples of Golgi dispersal and formation in non-neuronal cells: 1) A local de novo production from the ER; or 2) By fragmentation of the somatic Golgi followed by dispersal of remnant membranes that could serve as templates to rebuild Golgi stacks. In the present study we have tested which of these mechanisms, or alternative ones, are responsible for Golgi outpost formation and the role of elements of the fission machinery in such a process. Live cell imaging with spinning disk confocal microscopy reveals that Golgi outposts are generated from the somatic Golgi by a sequence of events involving: 1) Tubulation of a Golgi cisterna; 2) Elongation and penetration within a dendrite (deployment); 3) Tubule fission; and 4) Condensation of the fissioned tubule. Suppression of LIMK1, PKD1 or BARs reduce the number of Golgi outposts and induce Golgi deployment/tubulation without fission; by contrast, ectopic expression of these proteins stimulates Golgi outpost formation. This phenomenon can be stimulated by LPA, and FRET experiments show high RhoA activity in the Golgi area alter LPA stimulation.