Carey Fagerstrom

Molecular Requirements for Kinetochore-Associated Microtubule Formation in Mammalian Cells

In centrosome-containing cells, microtubules nucleated at centrosomes are thought to play a major role in spindle assembly. In addition, microtubule formation at kinetochores has also been observed, most recently under physiological conditions in live cells. The relative contributions of microtubule formation at kinetochores and centrosomes to spindle assembly, and their molecular requirements, remain incompletely understood. Using mammalian cells released from nocodazole-induced disassembly, we observed microtubule formation at centrosomes and at Bub1-positive sites on chromosomes. Kinetochore-associated microtubules rapidly coalesced into pole-like structures in a dynein-dependent manner. Microinjection of excess importin-beta or depletion of the Ran-dependent spindle assembly factor, TPX2, blocked kinetochore-associated microtubule formation, enhanced centrosome-associated microtubule formation, but did not prevent chromosome capture by centrosomal microtubules. Depletion of the chromosome passenger protein, survivin, reduced microtubule formation at kinetochores in an MCAK-dependent manner. Microtubule formation in cells depleted of Bub1 or Nuf2 was indistinguishable from that in controls. Our data demonstrate that microtubule assembly at centrosomes and kinetochores is kinetically distinct and differentially regulated. The presence of microtubules at kinetochores provides a mechanism to reconcile the time required for spindle assembly in vivo with that observed in computer simulations of search and capture.

Reorganization of the microtubule array in prophase/prometaphase requires cytoplasmic dynein-dependent microtubule transport

When mammalian somatic cells enter mitosis, a fundamental reorganization of the Mt cytoskeleton occurs that is characterized by the loss of the extensive interphase Mt array and the formation of a bipolar mitotic spindle. Microtubules in cells stably expressing GFP–α-tubulin were directly observed from prophase to just after nuclear envelope breakdown (NEBD) in early prometaphase. Our results demonstrate a transient stimulation of individual Mt dynamic turnover and the formation and inward motion of microtubule bundles in these cells. Motion of microtubule bundles was inhibited after antibody-mediated inhibition of cytoplasmic dynein/dynactin, but was not inhibited after inhibition of the kinesin-related motor Eg5 or myosin II. In metaphase cells, assembly of small foci of Mts was detected at sites distant from the spindle; these Mts were also moved inward. We propose that cytoplasmic dynein-dependent inward motion of Mts functions to remove Mts from the cytoplasm at prophase and from the peripheral cytoplasm through metaphase. The data demonstrate that dynamic astral Mts search the cytoplasm for other Mts, as well as chromosomes, in mitotic cells.

Quantification of microtubule nucleation, growth and dynamics in wound-edge cells

Mammalian cells develop a polarized morphology and migrate directionally into a wound in a monolayer culture. To understand how microtubules contribute to these processes, we used GFP-tubulin to measure dynamic instability and GFP-EB1, a protein that marks microtubule plus-ends, to measure microtubule growth events at the centrosome and cell periphery. Growth events at the centrosome, or nucleation, do not show directional bias, but are equivalent toward and away from the wound. Cells with two centrosomes nucleated approximately twice as many microtubules/minute as cells with one centrosome. The average number of growing microtubules per μm2 at the cell periphery is similar for leading and trailing edges and for cells containing one or two centrosomes. In contrast to microtubule growth, measurement of the parameters of microtubule dynamic instability demonstrate that microtubules in the trailing edge are more dynamic than those in the leading edge. Inhibition of Rho with C3 transferase had no detectable effect on microtubule dynamics in the leading edge, but stimulated microtubule turnover in the trailing edge. Our data demonstrate that in wound-edge cells, microtubule nucleation is non-polarized, in contrast to microtubule dynamic instability, which is highly polarized, and that factors in addition to Rho contribute to microtubule stabilization.

A conserved role for kinesin-5 in plant mitosis

The mitotic spindle of vascular plants is assembled and maintained by processes that remain poorly explored at a molecular level. Here, we report that AtKRP125c, one of four kinesin-5 motor proteins in arabidopsis, decorates microtubules throughout the cell cycle and appears to function in both interphase and mitosis. In a temperature-sensitive mutant, interphase cortical microtubules are disorganized at the restrictive temperature and mitotic spindles are massively disrupted, consistent with a defect in the stabilization of anti-parallel microtubules in the spindle midzone, as previously described in kinesin-5 mutants from animals and yeast. AtKRP125c introduced into mammalian epithelial cells by transfection decorates microtubules throughout the cell cycle but is unable to complement the loss of the endogenous kinesin-5 motor (Eg5). These results are among the first reports of any motor with a major role in anastral spindle structure in plants and demonstrate that the conservation of kinesin-5 motor function throughout eukaryotes extends to vascular plants.

Dynein Antagonizes Eg5 by Crosslinking and Sliding Antiparallel Microtubules

Mitotic spindle assembly requires the combined activity of various molecular motor proteins, including Eg5 and dynein. Together, these motors generate antagonistic forces during mammalian bipolar spindle assembly; what remains unknown, however, is how these motors are functionally coordinated such that antagonism is possible. Given that Eg5 generates an outward force by crosslinking and sliding apart antiparallel microtubules (MTs), we explored the possibility that dynein generates an inward force by likewise sliding antiparallel MTs. We reasoned that antiparallel overlap, and therefore the magnitude of a dynein-mediated force, would be inversely proportional to the initial distance between centrosomes. To capitalize on this relationship, we utilized a nocodazole washout assay to mimic spindle assembly. We found that Eg5 inhibition led to either monopolar or bipolar spindle formation, depending on whether centrosomes were initially separated by less than or greater than 5.5 microm, respectively. Mathematical modeling predicted this same spindle bistability in the absence of functional Eg5 and required dynein acting on antiparallel MTs to do so. Our results suggest that dynein functionally coordinates with Eg5 by crosslinking and sliding antiparallel MTs, a novel role for dynein within the framework of spindle assembly.

Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux

TPX2 is a spindle assembly factor that is required for MT assembly near chromosomes. Using photoactivation of fluorescence, we report that TPX2 is transported poleward in the half-spindle. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5, and to suppression of MT flux.

Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2

The kinesin Eg5 moves toward minus ends of astral microtubules in early mitosis, switching to plus-end motion in anaphase. Dynein is required for minus-end motion; depletion of TPX2 results in a switch to plus-end motion. On midzone microtubules, Eg5 moves in both directions. Our results explain the redistribution of Eg5 throughout mitosis.

Stable expression of fluorescently tagged proteins for studies of mitosis in mammalian cells.

Stable expression of fluorescently tagged proteins for studies of mitosis in mammalian cells

Fascetto Interacting Protein (FIP) Regulates Fascetto (PRC1) to Ensure Proper Cytokinesis and Ploidy

Cell division is critical for development, organ growth, and tissue repair. The later stages of cell division include the formation of the microtubule (MT)-rich central spindle in anaphase, which is required to properly define the cell equator, guide the assembly of the acto-myosin contractile ring, and ultimately ensure complete separation and isolation of the two daughter cells via abscission. Much is known about the molecular machinery that forms the central spindle, including proteins needed to generate the antiparallel overlapping interzonal MTs. One critical protein that has garnered great attention is Protein Regulator of Cytokinesis 1 (PRC1), or Fascetto (Feo) in Drosophila, which forms a homodimer to crosslink interzonal MTs, ensuring proper central spindle formation and cytokinesis. Here, we report on a new direct protein interactor and regulator of Feo we named Fascetto Interacting Protein (FIP). Loss of FIP results in a significant reduction in Feo localization, rapid disassembly of interzonal MTs, and several cytokinesis defects. Simultaneous reduction in Feo and FIP results in tumor-like, DNA-filled masses in the brain. In aggregate our data show that FIP functions upstream of, and acts directly on, Feo to ensure fully accurate cell division.