The Cytoskeleton of Diatoms

Abstract

The cytoskeleton is essential for many cellular functions such as cell motility, the control of cell shape and polarity, meiosis, cytokinesis, intracellular transport as well as endo- and exocytosis. The present study analyses the diatom actin and microtubule (MT) cytoskeleton from various sides: visualization through immunolabeling and reporter genes, as well as by in silico genomic analyses.<br /> This study presents for the first time immunolabeling of actin, α -tubulin, γ -tubulin as single label or in combinations in Craticula cuspidata. The actin cytoskeleton of this species is divers with a radially arranged fine cortical actin meshwork, a dens dynamic actin network in the deeper cytoplasm without obvious orientation and two thick prominent actin bundles parallel to the raphe. The actin drug, jasplakinolide allowed to gain insight into the dynamics of these structures. The results combined with previous publications indicate that the raphe bundles are highly dynamic structures recovering at a very fast speed after inhibitor treatment. Indicating, that actin filaments contributing to these bundles nucleate all along the raphe simultaneously rather than elongate unidirectional from the cell poles. Co-localization of γ -tubulin and MTs demonstrated the existence of multiple MT organizing centers (MTOCs) on the nuclear surface. MTs radiate out form the MTOCs into the periphery and further towards the cell poles, thus forming a highly symmetrical MT system.<br /> Cylindrotheca fusiformis was successfully transformed with the actin reporter, Lifeact-GFP. Expression of this construct allowed the visualization of all actin structures that were also labeled by immunofluorescence, such as the fine, cortical meshwork, the deeper cytoplasmic network and the thick bundles underneath the raphe. Now the experimental tool is available to address questions concerning the dynamics of the actin cytoskeleton and the connection of the actin cytoskeleton to the locomotion process of pennate diatoms. <br /> This study focuses also on an in silico genome inventory of actin, actin-related proteins (ARPs) and actin-binding proteins (ABPs) encoded in the genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. The comparative genomic and phylogenetic study revealed, that most diatoms possess only a single conventional actin and a small set of ARPs and ABPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. The genomes of the diatoms contain two structurally different homologues of Arp4 that might serve specific functions. All diatom species, examined here lack the ARP2/3-complex, which is essential in most eukaryotes for actin filament branching and plus- end dynamics. Diatoms encode a small set of ABPs, which should be efficient enough to regulate the disassembly of F-actin, the recycling of G-actin, as well as the capping of filaments and their anchoring to membranes. However, none of the sequenced representatives of the Bacillariophyta phylum encode for the essential actin regulating protein profilin. This is the first incidence of organisms not containing profilin. The hypothesis is put forward that disassembled ADP-actin is recycled back to ATP-actin by CAPs, though it remains unclear, how the activated actin is dissociated from CAPs to become available for polymerization at the filament plus-ends. Diatoms possess several multidomain variants of formin. All of them lack the profilin binding domain (FH1) suggesting that they are probably not capable of accelerating plus-end dynamics, a well studied function of formins in most other organisms. It therefore appears that diatoms have developed a novel, yet unknown way of filament growth and regulation of rapid filament elongation. This characteristic seems to have spread among the Stramenopiles, as most Stramenopila predominantly code for formins without a FH1 domain, but in diatoms it is most distinctly expressed.