The first insight into the in vivo
function of a microtubule severing enzyme came from studies in C. elegans
that identified two genes, MEI-1 and MEI-2 (the names for the C. elegans
katanin catalytic and regulatory subunits, respectively) as essential for the assembly of the acentriolar female meiotic spindle [8
]. Recent studies have brought fresh mechanistic insights into katanin’s role in building the C. elegans
meiotic spindle and established the importance of the microtubule severing enzymes katanin, spastin and fidgetin in additional fundamental cellular processes such as mitosis, cilia biogenesis, deflagellation and neurogenesis.
In C. elegans
oocytes, a katanin loss of function mutation results in failure to form a bipolar meiotic spindle [9
]. EM tomography of these mutant spindles revealed that they have fewer microtubules and that microtubules are longer than in wild-type embryos [10
]. Long microtubules are consistent with the long meiotic spindles and spindle shortening defects observed in a katanin partial loss of function mutant [3
], however it is still unclear why long microtubules would result in complete spindle assembly failure. Interestingly, the EM tomographic analysis showed a large decrease in the total microtubule mass in the katanin mutant meiotic spindle relative to wild-type, but only a small decrease in polymer mass was observed by light microscopy [3
]. Consistent with a role in regulating microtubule density, a synergistic loss of polymer mass was observed in a katanin γ-tubulin double mutant [3
]. These results suggest that microtubule severing during C. elegans
meiotic spindle assembly may increase polymer mass by generating shorter microtubules that can serve as seeds for nucleating new microtubules [11
]. By regulating the stability of the seeds (whether they depolymerize or not), microtubule density can be controlled. The stability of the seeds is most likely influenced by microtubule-associated factors and this will likely be an interesting area of future investigations.
In C. elegans
, persistence of katanin beyond meiosis results in damage to the mitotic spindle and chromosome segregation failure [13
], underscoring that cellular context is critical for the phenotypic outcomes of katanin severing. Thus, katanin levels are stringently controlled upon the transition from meiosis to mitosis via
two parallel proteolytic degradation pathways, the CUL-3 and MBK-2 pathways [14
]. This control mechanism is conserved in mammals [15
], however with a less dramatic outcome than in C. elegans
since katanin is still present in the mitotic spindles of vertebrates [17
], while absent from C. elegans
mitotic spindles. The significance of this difference is presently unclear.
Direct evidence for the role of severing in microtubule nucleation was uncovered in plants. Cortical microtubules in plants are arranged in a parallel array and are responsible for controlling the direction of cellulose deposition, and thereby cell shape. In vivo
imaging of GFP-tubulin revealed that many cortical microtubules nucleate off the wall of pre-existing microtubules at discrete 40° angles [18
]. The new microtubules are released from the branch point and then “move” predominantly by treadmilling [18
], which presumably allows them to arrange into a parallel array. In a katanin mutant (AT1G80350 in ), release of nascent microtubules from branch points is blocked [19
], explaining the lack of parallel microtubule organization, defective cellulose deposition and abnormal cell shape [20
]. Branch points contain γ-tubulin [21
] and interestingly, a point mutation in a γ-tu
omplex (γ-TuRC) subunit changes the angle at which the microtubule branches [19
], strongly indicating that its minus end is capped by a γ–TuRC. Release of microtubules from branch points by katanin likely removes the γ-TuRC from the minus end, a requirement for treadmilling-based movement of the freed microtubule ().
Our understanding of the cellular mechanism of katanin is more advanced in plants than in other systems because loss of function mutants are viable and katanin acts on interphase microtubules that are adequately spaced to allow direct observation of individual microtubules by light microscopy. In contrast, other subcellular structures where microtubule severing enzyme function is important, such as spindles, axons, dendrites and cilia, contain bundled microtubules with less than 200 nm spacing, thus currently precluding direct observation of individual microtubules by light microscopy.
The most complete functional analysis of microtubule severing enzymes has been in mitosis. In Drosophila
S2 spindles, spastin and fidgetin both localize to the centrosome and are required for microtubule minus end depolymerization, while katanin is required for plus end depolymerization [7
]. Spastin and fidgetin in mitotic S2 cells may uncap minus ends from γ-tubulin ring complexes as in the plant cortical cytoskeleton, but these events have not been directly observed at spindle poles due to the high microtubule density. It is worth noting that while fidgetin over-expression does cause microtubule disassembly in Drosophila
S2 cells [7
], purified fidgetin, unlike katanin and spastin, has not yet been shown to sever microtubules in vitro
. The C. elegans
fidgetin mutant shows defects consistent with a role during mitosis in the germline [22
] and mouse fidgetin mutants show an array of phenotypes [23
]; however, the specific cellular function of fidgetin has not been elucidated in these systems.
One of the most conserved roles for katanin is likely in the assembly and disassembly of cilia and flagella. Loss of function mutations in katanin subunit genes result in the assembly of flagella or cilia without a central microtubule pair in both Chlamydomonas
] and Tetrahymena
], two very distantly related organisms. Coincidentally, Arabidopsis
and C. elegans
, organisms with well-characterized loss of function katanin mutants, do not have cilia with a central pair at any time during their wild-type development. It is likely that a role for katanin in ciliogenesis in Drosophila
and mouse will be revealed when appropriate mutants are isolated. Recently, katanin has also been implicated in severing of basal bodies from the transition zone at the ends of resorbing flagella in Chlamydomonas
, a process that appears essential for the use of the basal bodies as mitotic spindle poles. Depletion of katanin is lethal but dividing cells with intact flagella accumulate in the population of dying cells [26
]. This is definitely an area that requires further investigation since resorption of cilia during mitosis likely occurs in most cells of the human body, but it is not known whether severing of the basal body is a required step in this process.
Consistent with the role of microtubule severing enzymes in the formation of complex, dense microtubule arrays such as those found in spindles or cilia, both spastin and katanin have functions in axonal elongation as well as branch formation in neurons. Spastin was originally identified as one of the most commonly mutated genes in hereditary spastic paraplegia [27
], a human neurodegenerative disease characterized by lower extremity weakness due to axonopathy. Disease mutations in humans either inactivate or downregulate spastin severing activity [4
]. In neurons, spastin localizes to the centrosome, synaptic boutons, points of new branch formation and its overexpression induces excessive branching [28
]. Loss of spastin function leads to sparse and disorganized microtubule arrays at the synaptic terminals of the neuromuscular junction in Drosophila
], as well as defects in dendritic arbor outgrowth and branching of a subclass of neurons characterized by their elaborate dendritic arbors [31
]. In zebra fish, spastin loss leads to a disorganized and sparse axonal microtubule array and impaired axonal outgrowth [32
]. Earlier work in hippocampal neurons also established a role for katanin in axonal elongation, presumably through the release of microtubules from the centrosome [33
Recently, a katanin-like protein (CG1193 in ) has been implicated in dendritic pruning in the Drosophila
nervous system [34
]. Specific sensory neurons initially have numerous dendrites that are eliminated during the transition from larvae to adult, reflecting a type of neural plasticity. In wild-type larvae, discontinuities or “breaks” in GFP-tubulin fluorescence within these dendrites precede visible breaks in a membrane marker, indicating that localized microtubule disassembly precedes severing of these dendrites. Both tissue specific RNAi and insertion mutations in a p60 katanin like protein cause a pronounced delay in the appearance of breaks in the tubulin fluorescence and a delay in dendrite severing [34
]. These results show a requirement for a katanin-like protein in localized microtubule disassembly during dendritic pruning; however, dendrites contain dense microtubule bundles [35
] making it very challenging to directly observe microtubule severing during dendritic pruning in wild-type neurons.