Chuanhai Fu

Crosslinkers and Motors Organize Dynamic Microtubules to Form Stable Bipolar Arrays in Fission Yeast

Microtubule (MT) nucleation not only occurs from centrosomes, but also in large part from dispersed nucleation sites. The subsequent sorting of short MTs into networks like the mitotic spindle requires molecular motors that laterally slide overlapping MTs and bundling proteins that statically connect MTs. How bundling proteins interfere with MT sliding is unclear. In bipolar MT bundles in fission yeast, we found that the bundler ase1p localized all along the length of antiparallel MTs, whereas the motor klp2p (kinesin-14) accumulated only at MT plus ends. Consequently, sliding forces could only overcome resistant bundling forces for short, newly nucleated MTs, which were transported to their correct position within bundles. Ase1p thus regulated sliding forces based on polarity and overlap length, and computer simulations showed these mechanisms to be sufficient to generate stable bipolar bundles. By combining motor and bundling proteins, cells can thus dynamically organize stable regions of overlap between cytoskeletal filaments.

Stabilization of PML nuclear localization by conjugation and oligomerization of SUMO-3

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells, resulting from a reciprocal chromosome translocation t (15;17). Here we show that the nuclear localization of PML is also regulated by SUMO-3, one of the three recently identified SUMO isoforms in human cells. SUMO-3 bears similar subcellular distribution to those of SUMO-1 and -2 in the interphase nuclear body, which is colocalized with PML protein. However, both SUMO-2 and -3 are also localized to nucleoli, a region lacking SUMO-1. Immunoprecipitated PML protein bears SUMO-3 moiety in a covalently modified form, supporting the notion that PML is conjugated by SUMO-3. To determine the functional relevance of SUMO-3 conjugation on PML molecular dynamics, we suppressed SUMO-3 protein expression using a siRNA-mediated approach. Depletion of SUMO-3 markedly reduced the number of PML-containing NBa and their integrity, which is rescued by exogenous expression of SUMO-3 but not SUMO-1 or SUMO-2. The specific requirement of SUMO-3 for PML nuclear localization is validated by expression of SUMO-3 conjugation defective mutant. Moreover, we demonstrate that oligomerization of SUMO-3 is required for PML retention in the nucleus. Taken together, our studies provide first line of evidence showing that SUMO-3 is essential for PML localization and offer novel insight into the pathobiochemistry of APL.

Phospho-regulated interaction between kinesin-6 Klp9p and microtubule bundler Ase1p promotes spindle elongation.

The spindle midzone-composed of antiparallel microtubules, microtubule-associated proteins (MAPs), and motors-is the structure responsible for microtubule organization and sliding during anaphase B. In general, MAPs and motors stabilize the midzone and motors produce sliding. We show that fission yeast kinesin-6 motor klp9p binds to the microtubule antiparallel bundler ase1p at the midzone at anaphase B onset. This interaction depends upon the phosphorylation states of klp9p and ase1p. The cyclin-dependent kinase cdc2p phosphorylates and its antagonist phosphatase clp1p dephosphorylates klp9p and ase1p to control the position and timing of klp9p-ase1p interaction. Failure of klp9p-ase1p binding leads to decreased spindle elongation velocity. The ase1p-mediated recruitment of klp9p to the midzone accelerates pole separation, as suggested by computer simulation. Our findings indicate that a phosphorylation switch controls the spatial-temporal interactions of motors and MAPs for proper anaphase B, and suggest a mechanism whereby a specific motor-MAP conformation enables efficient microtubule sliding.

PLK1 Phosphorylates Mitotic Centromere-associated Kinesin and Promotes Its Depolymerase Activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.

Mitotic Regulator Mis18β Interacts with and Specifies the Centromeric Assembly of Molecular Chaperone Holliday Junction Recognition Protein (HJURP)

Background: HJURP is a molecular chaperone essential for the deposition of the centromere marker CENP-A. Results: Mis18β binds with and specifies the centromere localization of HJURP. Conclusion: Mis18β governs centromere assembly via the Mis18β-HJURP-CENP-A axis. Significance: Our finding reveals a novel mechanism underlying CENP-A incorporation into the centromere. The centromere is essential for precise and equal segregation of the parental genome into two daughter cells during mitosis. CENP-A is a unique histone H3 variant conserved in eukaryotic centromeres. The assembly of CENP-A to the centromere is mediated by Holliday junction recognition protein (HJURP) in early G1 phase. However, it remains elusive how HJURP governs CENP-A incorporation into the centromere. Here we show that human HJURP directly binds to Mis18β, a component of the Mis18 complex conserved in the eukaryotic kingdom. A minimal region of HJURP for Mis18β binding was mapped to residues 437–460. Depletion of Mis18β by RNA interference dramatically impaired HJURP recruitment to the centromere, indicating the importance of Mis18β in HJURP loading. Interestingly, phosphorylation of HJURP by CDK1 weakens its interaction with Mis18β, consistent with the notion that assembly of CENP-A to the centromere is achieved after mitosis. Taken together, these data define a novel molecular mechanism underlying the temporal regulation of CENP-A incorporation into the centromere by accurate Mis18β-HJURP interaction.

PRC1 Cooperates with CLASP1 to Organize Central Spindle Plasticity in Mitosis

During cell division, chromosome segregation is governed by the interaction of spindle microtubules with the kinetochore. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, microtubule-bundling protein PRC1, and Aurora B kinase complex. However, the precise role of PRC1 in central spindle organization has remained elusive. Here we show that PRC1 recruits CLASP1 to the central spindle at early anaphase onset. CLASP1 belongs to a conserved microtubule-binding protein family that mediates the stabilization of overlapping microtubules of the central spindle. PRC1 physically interacts with CLASP1 and specifies its localization to the central spindle. Repression of CLASP1 leads to sister-chromatid bridges and depolymerization of spindle midzone microtubules. Disruption of PRC1-CLASP1 interaction by a membrane-permeable peptide abrogates accurate chromosome segregation, resulting in sister chromatid bridges. These findings reveal a key role for the PRC1-CLASP1 interaction in achieving a stable anti-parallel microtubule organization essential for faithful chromosome segregation. We propose that PRC1 forms a link between stabilization of CLASP1 association with central spindle microtubules and anti-parallel microtubule elongation.

Proteomic Identification and Functional Characterization of a Novel ARF6 GTPase-activating Protein, ACAP4

ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge of migrating cells. A key determinant of ARF6 function is the lifetime of the GTP-bound active state, which is orchestrated by GTPase-activating protein (GAP) and GTP-GDP exchanging factor. However, very little is known about the molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate ARF6 activity during cell migration, we performed a proteomic analysis of proteins selectively bound to active ARF6 using mass spectrometry and identified a novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids and contains two coiled coils, one pleckstrin homology domain, one GAP motif, and two ankyrin repeats. Our biochemical characterization demonstrated that ACAP4 has a phosphatidylinositol 4,5-bisphosphate-dependent GAP activity specific for ARF6. The co-localization of ACAP4 with ARF6 occurred in ruffling membranes formed upon AIF4 and epidermal growth factor stimulation. ACAP4 overexpression limited the recruitment of ARF6 to the membrane ruffles in the absence of epidermal growth factor stimulation. Expression of GTP hydrolysis-resistant ARF6Q67L resulted in accumulations of ACAP4 and ARF6 in the cytoplasmic membrane, suggesting that GTP hydrolysis is required for the ARF6-dependent membrane remodeling. Significantly the depletion of ACAP4 by small interfering RNA or inhibition of ARF6 GTP hydrolysis by overexpressing GAP-deficient ACAP4 suppressed ARF6-dependent cell migration in wound healing, demonstrating the importance of ACAP4 in cell migration. Thus, our study sheds new light on the biological function of ARF6-mediated cell migration.

Antagonistic spindle motors and MAPs regulate metaphase spindle length and chromosome segregation

Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at characteristic constant length [1-3]. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules (MTs) and their interactions with motors and MT-associated proteins (MAPs). Spindle length is further proposed to be important for chromosome segregation fidelity, as cells with shorter- or longer-than-normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force-balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic force contributors in the fission yeast metaphase spindle. We show that the spindle midzone proteins kinesin-5 cut7p and MT bundler ase1p contribute to outward-pushing forces and that the spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward-pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and in some combinations also partially rescued chromosome segregation defects.

Acetylation of Aurora B by TIP60 ensures accurate chromosomal segregation

Faithful chromosome segregation in mammalian cells requires the bi-orientation of sister chromatids which relies on sensing correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying cyclin-dependent kinase 1 (CDK1) activation that triggers mitotic entry is extensively studied, the regulatory mechanisms that couple CDK1-cyclin B activity to chromosome stability are not well understood. Here, we identified a signaling axis in which Aurora B activity is modulated by CDK1-cyclin B via acetyltransferase TIP60 (Tat-interactive protein 60 kDa) in human cell division. CDK1-cyclin B phosphorylated Ser90 of TIP60, which elicited TIP60-dependent acetylation of Aurora B and promoted accurate chromosome segregation in mitosis. Mechanistically, TIP60 acetylation of Aurora B at Lys215 protected the phosphorylation of its activation loop from PP2A reactivation-elicited dephosphorylation to ensure a robust, error-free metaphase-anaphase transition. These findings delineated a conserved signaling cascade that integrates protein phosphorylation and acetylation to cell cycle progression for maintenance of genomic stability.

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

Significance The spindle assembly checkpoint (SAC) works as a surveillance mechanism to ensure accurate segregation of genetic materials during cell division. Protein kinase monopolar spindle 1 (Mps1) plays a key role in SAC, but the mechanism of Mps1 action in chromosome segregation remains elusive. In this study, we identified a previously undefined structural determinant of Mps1 named “IRK” (internal region for kinetochore localization) and demonstrated its functional importance in accurate kinetochore–microtubule attachment. Mechanistically, a dynamic hierarchical interaction between Mps1 and the nuclear division cycle 80 complex (Ndc80C) orchestrates accurate mitosis, because persistent association of inactive Mps1 with Ndc80C via the IRK perturbs correct kinetochore–microtubule attachment. Our results provide a new mechanistic insight into the spatiotemporal dynamics of Mps1 activity at the kinetochore in mitosis. The spindle assembly checkpoint (SAC) is a conserved signaling pathway that monitors faithful chromosome segregation during mitosis. As a core component of SAC, the evolutionarily conserved kinase monopolar spindle 1 (Mps1) has been implicated in regulating chromosome alignment, but the underlying molecular mechanism remains unclear. Our molecular delineation of Mps1 activity in SAC led to discovery of a previously unidentified structural determinant underlying Mps1 function at the kinetochores. Here, we show that Mps1 contains an internal region for kinetochore localization (IRK) adjacent to the tetratricopeptide repeat domain. Importantly, the IRK region determines the kinetochore localization of inactive Mps1, and an accumulation of inactive Mps1 perturbs accurate chromosome alignment and mitotic progression. Mechanistically, the IRK region binds to the nuclear division cycle 80 complex (Ndc80C), and accumulation of inactive Mps1 at the kinetochores prevents a dynamic interaction between Ndc80C and spindle microtubules (MTs), resulting in an aberrant kinetochore attachment. Thus, our results present a previously undefined mechanism by which Mps1 functions in chromosome alignment by orchestrating Ndc80C–MT interactions and highlight the importance of the precise spatiotemporal regulation of Mps1 kinase activity and kinetochore localization in accurate mitotic progression.