Biology subjects

Maresca, T. J.

Publications and source records attributed to Maresca, T. J..

6 recordsLinked to original sources

bioRxiv2024

Proximity-based activation of AURORA A by MPS1 potentiates error correction

Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

cell biology↗

bioRxiv2023

Human dynein-dynactin is a fast processive motor in living cells

Minus-end directed transport along microtubules in eukaryotes is primarily mediated by cytoplasmic dynein and its cofactor dynactin. Significant advances have been made in recent years characterizing human dynein-dynactin structure and function using in vitro assays, however, there is limited knowledge about the motile properties and functional organization of dynein-dynactin in living human cells. Total internal reflection fluorescence microscopy (TIRFM) of CRISPR-engineered human cells is employed here to visualize fluorescently tagged dynein heavy chain (DHC) and p50 with high spatio-temporal resolution. We find that p50 and DHC exhibit indistinguishable motility properties in their velocities, run lengths, and run times. The dynein-dynactin complexes are fast ([~]1.2 m/s) and run for several microns ([~]2.7 m). Quantification of the fluorescence intensities of motile puncta reveals that dynein-dynactin runs are mediated by at least one DHC dimer while the velocity is consistent with that measured for double dynein (two DHC dimers) complexes in vitro.

cell biology↗

bioRxiv2023

An intrinsically disordered kinetochore protein coordinates mechanical regulation of chromosome segregation by dynein

Kinetochores connect chromosomes and spindle microtubules to maintain genomic integrity through cell division. Crosstalk between the minus-end directed motor dynein and kinetochore-microtubule attachment factors promotes accurate chromosome segregation through a poorly understood pathway. Here we identify a physical linkage between the intrinsically disordered protein Spc105 (KNL1 orthologue) and dynein using an optogenetic oligomerization assay. Core pools of the checkpoint protein BubR1 and the adaptor complex RZZ mediate the connection of Spc105 to dynein. Furthermore, a minimal segment of Spc105 that contains regions with a propensity to multimerize and binding motifs for Bub1 and BubR1 is sufficient to functionally link Spc105 to RZZ and dynein. Deletion of the minimal region from Spc105 reduces recruitment of its binding partners to bioriented kinetochores and causes chromosome mis-segregation. Restoration of normal chromosome segregation and localization of BubR1 and RZZ requires both protein binding motifs and higher-order oligomerization of Spc105. Together, our results reveal that higher-order multimerization of Spc105 is required to recruit a core pool of RZZ that modulates microtubule attachment stability to promote accurate chromosome segregation.

cell biology↗

bioRxiv2019

Direct observation of branching MT nucleation in living animal cells.

Centrosome-mediated microtubule (MT) nucleation has been well-characterized; however, numerous non-centrosomal MT nucleation mechanisms exist. The branching MT nucleation pathway envisages that the {gamma}-tubulin ring complex ({gamma}-TuRC) is recruited to MTs by the augmin complex to initiate nucleation of new MTs. While the pathway is well-conserved at a molecular and functional level, branching MT nucleation by core constituents has never been directly observed in animal cells. Here, multi-color TIRF microscopy was applied to visualize and quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells. The stereotypical branching nucleation event entailed augmin first binding to a \"mother\" MT, recruitment of {gamma}-TuRC after 16s, followed by nucleation 15s later of a \"daughter\" MT at a 36{degrees} branch angle. Daughters typically remained attached throughout their ~40s lifetime unless the mother depolymerized past the branch point. Assembly of branched MT arrays, which did not require D-TPX2 (Drosophila TPX2) or evident regulation by a RanGTP gradient, enhanced localized RhoA activation during cytokinesis.

cell biology↗

bioRxiv2018

Development of a genetically encoded sensor for endogenous CaMKII activity

CaMKII is a crucial oligomeric enzyme in neuronal and cardiac signaling, fertilization and immunity. Here, we report the construction of a novel, substrate-based, genetically-encoded sensor for CaMKII activity, FRESCA (FRET-based Sensor for CaMKII Activity). Currently, there is one biosensor for CaMKII activity, Camui, which contains CaMKII. FRESCA allows us to measure all endogenous CaMKII variants, while Camui can track a single variant. Since there are ~40 CaMKII variants, using FRESCA to measure aggregate activity allows a fresh perspective on CaMKII activity. We show, using live-cell imaging, FRESCA response is concurrent with Ca2+ rises in HEK293T cells and mouse eggs. In eggs, we stimulate oscillatory patterns of Ca2+ and observe the differential responses of FRESCA and Camui. Our results implicate an important role for the variable linker region in CaMKII, which tunes its activation. FRESCA will be a transformative tool for studies in neurons, cardiomyocytes and other CaMKII-containing cells.

biochemistry↗

bioRxiv2018

Microtubule plus-tips act as signaling hubs for positioning the cleavage furrow during cytokinesis.

Cell division in animal cells culminates with the formation of a contractile ring that divides the cytosol through formation of a cleavage furrow. Microtubules (MTs) are essential for furrow positioning, but the molecular nature of MT-derived spatial signals is unresolved. In this study essential cytokinesis regulators (the centralspindlin complex, aurora B kinase (ABK), and polo kinase) were visualized in Drosophila melanogaster (Dm) cells and found localize to and track MT plus-ends during cytokinesis. The RhoA GEF Pebble (Dm ECT2) did not robustly tip-track but became enriched at MT plus-tips rapidly following cortical contact resulting in RhoA activation and enrichment of myosin-regulatory light chain. Abrogation of cytokinesis regulator tip-tracking by EB1 depletion or deletion of a novel EB1-interaction motif (hxxPTxh) in the centralspindlin component RacGAP50C resulted in higher incidences of cytokinesis failure. We propose that EB1-dependent, MT plus-tip-based signaling hubs recruit cortical Dm ECT2 upon contact to locally activate RhoA.

cell biology↗