Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1

Abstract

It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

Centrosome Positioning in Dictyostelium: Moving beyond Microtubule Tip Dynamics

“…Disruption of either Kinesin-8 or Kinesin-4 isoforms (Ddkif10, Ddkif8, respectively) was sufficient to abrogate the dynein perturbation and in part, perturb the radial character of the MT array [ 20 ]. Deletion of Kif8 or the MT associated protein, Ase1A was also sufficient to alter the radial character of the MT arrays ( Figure 2 ) [ 9 , 20 ].…”

“…This observation suggests a mechanism that senses and reduces interaction between MT arrays, beyond that provided by simple motor protein and tip dynamics. To address this possibility, we targeted a MT effector protein family known to interact with MTs of opposite polarity (MAP65/Ase1/PRC1) [ 9 ]. In many animal cells, PRC1/Ase1 functions to organize MT overlap in the central spindle region during division and as anticipated, DdAse1A performs a similar mitotic function in D. discoideum .…”

“…On entry to mitosis, the cytoplasmic MT array dissolves, the centrosome begins to duplicate via longitudinal splitting of the core structure, inserts into an opening of the nuclear envelope, and organizes a compact intranuclear spindle similar in morphology to those seen in fungi ( Figure 1 ) [ 7 ]. Pole separation during anaphase is extensive, facilitated by pulling forces acting on the spindle astral MTs and tempered by Kinesin-5 action in the spindle midzone [ 8 , 9 ]. As mitosis nears completion, astral MTs grow and the centrosome is extruded back into the cytoplasm to complete the cycle.…”

13 Plus 1: A 30-Year Perspective on Microtubule-Based Motility in Dictyostelium

“…This function may reflect an evolutionary difference in spindle elongation mechanisms, where here and in some other organisms, astral pulling forces dominate over MT pushing for anaphase-B elongation [46]. The rapid pole separation seen in D. discoideum cells that lack a proper spindle overlap zone (e.g., [47]) suggests that cortical dynein pulling forces may be the primary driver for spindle elongation, and thus DdKif13 may be fine-tuned for a governor-type activity in the spindle midzone. In support of this idea, it is interesting to note that the primary antagonist of Kinesin-5 motors in animal-cell spindle assemblies is Kinesin-14; these two motors typically counterbalance actions to maintain spindle bipolarity [48,49].…”

CDK5RAP2 Is an Essential Scaffolding Protein of the Corona of the Dictyostelium Centrosome

“…Next, the core structure inserts into a fenestra opening in the nuclear envelope and the central layer disappears, while the formerly outer layers separate from each other within the nuclear envelope and represent the two mitotic spindle poles. The latter organize a central spindle under requirement of the microtubule crosslinker Ase1 [ 15 ]. Starting with the transition to anaphase, the plaque-like mitotic centrosomes fold back onto themselves, in a way that the microtubule-nucleating, formerly inner surfaces become oriented towards the outside.…”