Moonlighting of mitotic regulators in cilium disassembly

Correct timing of cellular processes is essential during embryological development and to maintain the balance between healthy proliferation and tumour formation. Assembly and disassembly of the primary cilium, the cell’s sensory signalling organelle, are linked to cell cycle timing in the same manner as spindle pole assembly and chromosome segregation. Mitotic processes, ciliary assembly, and ciliary disassembly depend on the centrioles as microtubule-organizing centres (MTOC) to regulate polymerizing and depolymerizing microtubules. Subsequently, other functional protein modules are gathered to potentiate specific protein–protein interactions. In this review, we show that a significant subset of key mitotic regulator proteins is moonlighting at the cilium, among which PLK1, AURKA, CDC20, and their regulators. Although ciliary assembly defects are linked to a variety of ciliopathies, ciliary disassembly defects are more often linked to brain development and tumour formation. Acquiring a better understanding of the overlap in regulators of ciliary disassembly and mitosis is essential in finding therapeutic targets for the different diseases and types of tumours associated with these regulators.

Loss of Kinesin-8 improves the robustness of the acentrosomal spindle

Chromosome segregation in female meiosis is inherently error-prone, among other reasons, because the acentrosomal spindle assembles and segregates chromosomes without the major microtubule-organizing centres in eukaryotes, the centrosomes, which causes high rate of aneuploidy. The molecular basis underlying formation of acentrosomal spindles is not as well-understood as that of centrosomal spindles and, consequently, strategies to improve spindle robustness are difficult to address. Recently, we noticed during fission yeast meiosis the formation of unexpected microtubules arrays, independent of the spindle pole bodies (yeast centrosome equivalent), with ability to segregate chromosomes. Here, we establish such microtubules formation as bonafide self-assembled spindles that depend on the canonical microtubule crosslinker Ase1/PRC1, share similar structural polarity and harbour the microtubule polymerase Alp14/XMAP215, while being independent of conventional γ-tubulin-mediated nucleation mechanisms. Remarkably, acentrosomal spindle robustness was reinforced by deletion of the Klp6/Kinesin-8, which, consequently, led to a reduced meiotic aneuploidy rate. Our results enlighten the molecular basis of acentrosomal meiosis, a crucial event in understanding gametogenesis.

Coordinated crosstalk between microtubules and actin by a spectraplakin regulates lumen formation and branching

SUMMARYThe establishment of branched structures by single cells involves complex cytoskeletal remodelling events. In Drosophila, epithelial tracheal system terminal cells (TCs) and dendritic arborisation neurons are models for these subcellular branching processes. During tracheal embryonic development, the generation of subcellular branches is characterized by extensive remodelling of the microtubule (MT) network and actin cytoskeleton, followed by vesicular transport and membrane dynamics. We have previously shown that centrosomes are key players in the initiation of subcellular lumen formation where they act as microtubule organizing centres (MTOCs). However, not much is known on the events that lead to the growth of these subcellular luminal branches or what makes them progress through a particular trajectory within the cytoplasm of the TC. Here, we have identified that the spectraplakin Short-stop (Shot) promotes the crosstalk between MTs and actin, which leads to the extension and guidance of the subcellular lumen within the TC cytoplasm. Shot is enriched in cells undergoing the initial steps of subcellular branching as a direct response to FGF signalling. An excess of Shot induces ectopic acentrosomal branching points in the embryonic and larval tracheal TC leading to cells with extra subcellular lumina. These data provide the first evidence for a role for spectraplakins in subcellular lumen formation and branching.

A centriolar FGR1 oncogene partner-like protein required for paraflagellar rod assembly, but not axoneme assembly in African trypanosomes

Proteins of the FGR1 oncogene partner (or FOP) family are found at microtubule organizing centres (MTOCs) including, in flagellate eukaryotes, the centriole or flagellar basal body from which the axoneme extends. We report conservation of FOP family proteins, Tb FOPL and Tb OFD1, in the evolutionarily divergent sleeping sickness parasite Trypanosoma brucei , showing (in contrast with mammalian cells, where FOP is essential for flagellum assembly) depletion of a trypanosome FOP homologue, Tb FOPL, affects neither axoneme nor flagellum elongation. Instead, Tb FOPL depletion causes catastrophic failure in assembly of a lineage-specific, extra-axonemal structure, the paraflagellar rod (PFR). That depletion of centriolar Tb FOPL causes failure in PFR assembly is surprising because PFR nucleation commences approximately 2 µm distal from the basal body. When over-expressed with a C-terminal myc-epitope, Tb FOPL was also observed at mitotic spindle poles. Little is known about bi-polar spindle assembly during closed trypanosome mitosis, but indication of a possible additional MTOC function for Tb FOPL parallels MTOC localization of FOP-like protein TONNEAU1 in acentriolar plants. More generally, our functional analysis of Tb FOPL emphasizes significant differences in evolutionary cell biology trajectories of FOP-family proteins. We discuss how at the molecular level FOP homologues may contribute to flagellum assembly and function in diverse flagellates.

Assembly and regulation of γ-tubulin complexes

Microtubules are major constituents of the cytoskeleton in all eukaryotic cells. They are essential for chromosome segregation during cell division, for directional intracellular transport and for building specialized cellular structures such as cilia or flagella. Their assembly has to be controlled spatially and temporally. For this, the cell uses multiprotein complexes containing γ-tubulin. γ-Tubulin has been found in two different types of complexes, γ-tubulin small complexes and γ-tubulin ring complexes. Binding to adaptors and activator proteins transforms these complexes into structural templates that drive the nucleation of new microtubules in a highly controlled manner. This review discusses recent advances on the mechanisms of assembly, recruitment and activation of γ-tubulin complexes at microtubule-organizing centres.

Ninein is essential for apico-basal microtubule formation and CLIP-170 facilitates its redeployment to non-centrosomal microtubule organizing centres

Differentiation of columnar epithelial cells involves a dramatic reorganization of the microtubules (MTs) and centrosomal components into an apico-basal array no longer anchored at the centrosome. Instead, the minus-ends of the MTs become anchored at apical non-centrosomal microtubule organizing centres (n-MTOCs). Formation of n-MTOCs is critical as they determine the spatial organization of MTs, which in turn influences cell shape and function. However, how they are formed is poorly understood. We have previously shown that the centrosomal anchoring protein ninein is released from the centrosome, moves in a microtubule-dependent manner and accumulates at n-MTOCs during epithelial differentiation. Here, we report using depletion and knockout (KO) approaches that ninein expression is essential for apico-basal array formation and epithelial elongation and that CLIP-170 is required for its redeployment to n-MTOCs. Functional inhibition also revealed that IQGAP1 and active Rac1 coordinate with CLIP-170 to facilitate microtubule plus-end cortical targeting and ninein redeployment. Intestinal tissue and in vitro organoids from the Clip1/Clip2 double KO mouse with deletions in the genes encoding CLIP-170 and CLIP-115, respectively, confirmed requirement of CLIP-170 for ninein recruitment to n-MTOCs, with possible compensation by other anchoring factors such as p150 Glued and CAMSAP2 ensuring apico-basal microtubule formation despite loss of ninein at n-MTOCs.

Exotic mitotic mechanisms

The emergence of eukaryotes around two billion years ago provided new challenges for the chromosome segregation machineries: the physical separation of multiple large and linear chromosomes from the microtubule-organizing centres by the nuclear envelope. In this review, we set out the diverse solutions that eukaryotic cells use to solve this problem, and show how stepping away from ‘mainstream’ mitosis can teach us much about the mechanisms and mechanics that can drive chromosome segregation. We discuss the evidence for a close functional and physical relationship between membranes, nuclear pores and kinetochores in generating the forces necessary for chromosome segregation during mitosis.