The Cytoskeleton of Diatoms : Structural and Genomic Analysis

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. 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. 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. 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

Cell scientist to watch – Charlotte Aumeier

Charlotte Aumeier is an Assistant Professor of Biochemistry at the University of Geneva, Switzerland. After completing her PhD at the University of Bonn, Germany, studying the cytoskeleton of diatoms, Charlotte moved to the University of Grenoble, France, for a postdoc, where she became hooked on microtubules. In 2018, she established her own lab in Geneva, where she and her team work to uncover the dynamics and regulatory mechanisms of microtubule networks using a variety of approaches, including synthetic biology and non-standard model organisms. We spoke with Charlotte over Zoom to learn more about her work, her love of diatoms and how she views some of the biggest challenges of her career as her biggest advantages as a scientist.</p

Regulation of the microtubule network; <i>the shaft matters!</i>

In cells, the microtubule network continually assembles and disassembles. The regulation of microtubule growth or shortening has almost exclusively been studied at their dynamic ends. However, microtubules are dynamic all along their entire shaft. A dynamic shaft increases the lifetime and length of a microtubule by reducing the shortening phases and promoting its regrowth. Here, we discuss how shaft dynamics can regulate microtubule network organization, intracellular transport, and polarization of the network

The effect of motor-induced shaft dynamics on microtubule stability and length

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depends on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in presence of kinesin-1 molecular motors. Although the average microtubule dynamics can be captured by an effective rescue rate, the dynamics of individual microtubules differs dramatically when rescue occurs only at exchange sites. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network

Microtubule damage shapes the acetylation gradient

The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen

Microtubule damage shapes the acetylation gradient

Abstract The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen

A liquid +TIP-network drives microtubule dynamics through tubulin condensation

Tubulin dimers assemble into a dynamic microtubule network throughout the cell. Microtubule dynamics and network organization must be precisely tuned for the microtubule cytoskeleton to regulate a dazzling array of dynamic cell behaviors. Since tubulin concentration determines microtubule growth, we studied here a novel regulatory mechanism of microtubule dynamics: local tubulin condensation. We discovered that two microtubule tip-binding proteins, CLIP-170 and EB3, undergo phase separation and form an EB3/CLIP-170 droplet at the growing microtubule tip. We prove that this +TIP-droplet has the capacity to locally condense tubulin. This process of tubulin co-condensation is spatially initiated at the microtubule tip and temporally regulated to occur only when there is tip growth. Tubulin condensation at the growing microtubule tip increases growth speeds three-fold and strongly reduces depolymerization events. With this work we establish a new mechanism to regulate microtubule dynamics by enrichment of tubulin at strategically important locations: the growing microtubule tips

Phase separation of +TIP networks regulates microtubule dynamics

Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here, we show that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid networks. When CLIP-170 and EB3 act together, the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells, these liquid networks can concentrate tubulin. In vitro, in the presence of microtubules, phase separation of EB3/CLIP-170 can enrich tubulin all along the microtubule. In this condition, microtubule growth speed increases up to twofold and the frequency of depolymerization events are strongly reduced compared to conditions in which there is no phase separation. Our data show that phase separation of EB3/CLIP-170 adds an additional layer of regulation to the control of microtubule growth dynamics.</p

Two-color in vitro assay to visualize and quantify microtubule shaft dynamics

Microtubules are dynamic polymers where tubulin exchanges not only at the ends but also all along the microtubule shaft. In vitro reconstitutions are a vital approach to study microtubule tip dynamics, while direct observation of shaft dynamics is challenging. Here, we describe a dual-color in vitro assay to visualize microtubule shaft dynamics using purified, labeled bovine brain tubulin. With this assay, we can quantitatively address how proteins or small molecules impact the dynamics at the microtubule shaft. For complete details on the use and execution of this protocol, please refer to Andreu-Carbó et al. (2022)