Jérémie Gaillard

Functional evidence for in vitro microtubule severing by the plant katanin homologue

Temporal and spatial assembly of microtubules in plant cells depends mainly on the activity of microtubule-interacting proteins, which either stabilize, destabilize or translocate microtubules. Recent data have revealed that the thale cress (Arabidopsis thaliana) contains a protein related to the p60 catalytic subunit of animal katanin, a microtubule-severing protein. However, effects of the plant p60 on microtubule assembly are not known. We report the first functional evidence that the recombinant A. thaliana p60 katanin subunit, Atp60, binds to microtubules and severs them in an ATP-dependent manner in vitro. ATPase activity of Atp60 is stimulated by low tubulin/katanin ratios, and is inhibited at higher ratios. Considering its properties in vitro, several functions of Atp60 in vivo are discussed.

Differential interactions of the formins INF2, mDia1, and mDia2 with microtubules

Three mammalian formins, although binding microtubules with high affinity, differ dramatically in their microtubule-binding mechanisms. In addition, the ability of one formin (mDia2) to bind actin is strongly inhibited by microtubules, whereas the ability of another formin (INF2) to bind microtubules is strongly inhibited by actin monomers.

Microtubules acquire resistance from mechanical breakage through intralumenal acetylation

Acetylation keeps microtubules strong Cells need microtubules for intracellular transport and to avoid being crushed. On investigating microtubule breakage in live fibroblasts, Xu et al. found that if they were not acetylated, long-lived microtubules underwent frequent rupture after buckling. Acetylation makes microtubules more mechanically stable, facilitates sliding between filaments, and makes the lattice more plastic. Science, this issue p. 328 Control of microtubule mechanics by acetylation is described. Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.

The centrosome is an actin-organizing centre

Microtubules and actin filaments are the two main cytoskeleton networks supporting intracellular architecture and cell polarity. The centrosome nucleates and anchors microtubules and is therefore considered to be the main microtubule-organizing centre. However, recurring, yet unexplained, observations have pointed towards a connection between the centrosome and actin filaments. Here we have used isolated centrosomes to demonstrate that the centrosome can directly promote actin-filament assembly. A cloud of centrosome-associated actin filaments could be identified in living cells as well. Actin-filament nucleation at the centrosome was mediated by the nucleation-promoting factor WASH in combination with the Arp2/3 complex. Pericentriolar material 1 (PCM1) seemed to modulate the centrosomal actin network by regulating Arp2/3 complex and WASH recruitment to the centrosome. Hence, our results reveal an additional facet of the centrosome as an intracellular organizer and provide mechanistic insights into how the centrosome can function as an actin-filament-organizing centre.

MAP65/Ase1 promote microtubule flexibility

Two microtubule cross-linkers of the major MAP65/PRC1/Ase1 family are found to modify the mechanical properties of dynamic microtubules (e.g., decrease the flexural rigidity of microtubules). This finding points to a role for these proteins in the formation of specific microtubule arrays in eukaryotic cells.

Arabidopsis Kinetochore Fiber-Associated MAP65-4 Cross-Links Microtubules and Promotes Microtubule Bundle Elongation

This study shows that Arabidopsis MAP65-4 associates with the forming spindle and kinetochore fibers during mitosis. In vitro, MAP65-4 induces microtubule (MT) bundling and modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, which results in the progressive lengthening of MT bundles. The acentrosomal plant mitotic spindle is uniquely structured in that it lacks opposing centrosomes at its poles and is equipped with a connective preprophase band that regulates the spatial framework for spindle orientation and mobility. These features are supported by specialized microtubule-associated proteins and motors. Here, we show that Arabidopsis thaliana MAP65-4, a non-motor microtubule associated protein (MAP) that belongs to the evolutionarily conserved MAP65 family, specifically associates with the forming mitotic spindle during prophase and with the kinetochore fibers from prometaphase to the end of anaphase. In vitro, MAP65-4 induces microtubule (MT) bundling through the formation of cross-bridges between adjacent MTs both in polar and antipolar orientations. The association of MAP65-4 with an MT bundle is concomitant with its elongation. Furthermore, MAP65-4 modulates the MT dynamic instability parameters of individual MTs within a bundle, mainly by decreasing the frequency of catastrophes and increasing the frequency of rescue events, and thereby supports the progressive lengthening of MT bundles over time. These properties are in line with its role of initiating kinetochore fibers during prospindle formation.

Self-repair promotes microtubule rescue

The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events can arrest the depolymerization and restore microtubule elongation. However, the origin of these rescue events remains unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints.

Quantification of MAP and molecular motor activities on geometrically controlled microtubule networks

The spatial organization of the microtubule (MT) network directs cell polarity and mitosis. It is finely regulated by hundreds of different types of microtubule‐associated proteins and molecular motors whose specific functions are difficult to investigate directly in cells. Here, we have investigated their functions using geometrically controlled MT networks in vitro in cell‐free assay. This was achieved by developing a new method to spatially define MT nucleation using MT microseeds adsorbed on a micropatterned glass substrate. This method could be used to control MT growth and the induction of complex MT networks. We selected the interaction of two radial arrays of dynamic and polarized MTs to analyze the formation of the central antiparallel MT bundle. We investigated the effects of the MT cross‐linker anaphase spindle elongation 1 (Ase1) and the kinesin motor Klp2, which are known to regulate MT organization in the spindle midzone. We thus identified the respective roles of each protein and revealed their synergy on the establishment of stable antiparallel MT bundles by quantifying MT interactions over hundreds of comparable MT networks.

Actin nucleation at the centrosome controls lymphocyte polarity

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome—regulated by the availability of the Arp2/3 complex—determines its capacity to polarize in response to external stimuli.

Plant katanin, a microtubule severing protein

Microtubules are highly dynamic polymers built from the addition and loss of tubulin dimers at their ends. Microtubule dynamics are essential to achieve fundamental cellular processes such as cell division. In vivo, microtubule assembly is tightly regulated. This regulation mainly results from the activity of microtubule effectors that interact with microtubules or tubulin dimers. There are two main types of microtubule effectors, those that favour the polymerised state of microtubules (mostly MAPs), and those that promote microtubule depolymerisation. Katanin is the best described microtubule-severing protein. Animal katanin is a hetero-dimer protein, composed of a catalytic subunit of 60 kDa (P60) and a regulatory subunit of 80 kDa (P80). P60 is an AAA (ATPases associated with various cellular activities) protein which hydrolyses ATP in a microtubule-dependent manner and is sufficient to sever microtubules in vitro (McNally et al., 2000). In animal cells, katanin is thought to be involved in the release of microtubules from centrosomes and the regulation of the number of microtubule ends in the spindle (Buster et al., 2002). In higher plants, no homologue of the P80 katanin regulatory subunit has been described so far. Recently two Arabidopsis thaliana mutants mutated in a gene showing significant homologies with the animal P60 catalytic katanin subunit have been described (Bichet et al., 2001; Burk et al., 2001; for a description of katanin mutants, see the abstract of D. Bouchez on the BOTERO mutant). Surprisingly, the microtubule cytoskeleton of these mutants has only very subtle defects, and cells seem to divide normally. Molecular effects of the plant P60 on microtubule assembly were not known. To study microtubule destabilization in plant cells, we cloned the cDNA encoding for the P60 orthologue in A. thaliana (AtP60) and functionally characterized the properties of a recombinant His-tagged AtP60 (Stoppin-Mellet et al., 2002). Using both video-microscopy assays and spectrofluorimetry, we showed for the first time that HisAtP60 can sever microtubules in vitro in the presence of ATP. His-AtP60 directly interacts with microtubules in co-sedimentation assays. In the presence of microtubules, the ATPase activity of AtP60 was stimulated in a non-hyperbolic way. The basal ATPase activity of AtP60 (calculated at one molecule ATP/AtP60/sec) was stimulated up to six times at low AtP60/tubulin molar ratio (maximum at 0.04), and inhibited at higher ratios. AtP60 is the first plant protein shown to fragment microtubules. The characterization of the functional domains of AtP60 is now under way, both in vitro and in vivo.