Abstract
Acentriolar microtubule organizing centers (aMTOCs) are formed during meiosis and mitosis in several cell types, but their function and assembly mechanism is unclear. Importantly, aMTOCs can be overactive in cancer cells, enhancing multipolar spindle formation, merotelic kinetochore attachment and aneuploidy. Here we show that aMTOCs can form in acentriolar Drosophila somatic cells in vivo via an assembly pathway that depends on Asl, Cnn and, to a lesser extent, Spd-2—the same proteins that appear to drive mitotic centrosome assembly in flies. This finding enabled us to ablate aMTOC formation in acentriolar cells, and so perform a detailed genetic analysis of the contribution of aMTOCs to acentriolar mitotic spindle formation. Here we show that although aMTOCs can nucleate microtubules, they do not detectably increase the efficiency of acentriolar spindle assembly in somatic fly cells. We find that they are required, however, for robust microtubule array assembly in cells without centrioles that also lack microtubule nucleation from around the chromatin. Importantly, aMTOCs are also essential for dynein-dependent acentriolar spindle pole focusing and for robust cell proliferation in the absence of centrioles and HSET/Ncd (a kinesin essential for acentriolar spindle pole focusing in many systems). We propose an updated model for acentriolar spindle pole coalescence by the molecular motors Ncd/HSET and dynein in conjunction with aMTOCs.
Highlights
Centrosomes are the major microtubule organizing centers (MTOCs) in many cells, and they consist of a pair of centrioles surrounded by a cloud of pericentriolar material (PCM), which contains proteins that nucleate and stabilize microtubules (MTs)
We identified the pathway of Acentriolar microtubule organizing centers (aMTOCs) formation in Drosophila, which enabled us to perturb their formation in order to study their role during spindle formation
We found that aMTOCs consistently form in ~50– 60% of mitotic fly somatic brain cells that lack centrioles, and that aMTOC assembly depends on the same proteins that are required to drive mitotic PCM assembly around the centrioles: Asl, Spd-2 and Cnn [37,38,39,40,41,42,43,44,45]
Summary
Centrosomes are the major microtubule organizing centers (MTOCs) in many cells, and they consist of a pair of centrioles surrounded by a cloud of pericentriolar material (PCM), which contains proteins that nucleate and stabilize microtubules (MTs). MT assembly is induced around the chromatin and the plus ends of these MTs are captured by the kinetochores and continue to grow from there in a MT bundle [5,6,7,8] This leads to the formation of kinetochore fibers (K fibers) with minus ends that are pushed away from the kinetochores. These K fibers coalesce into a bipolar spindle together with astral MTs emanating from the centrosomes in a mechanism involving three distinct steps: First, K fibers become crosslinked by the kinesin-14 Ncd/HSET. The augmin complex is required for spindle MT amplification by nucleating MTs that branch off the sides of existing MTs, which increases the density of MTs within the mitotic spindle [10,11,12,13,14,15]
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