Biology subjects

Gama, J. B.

Publications and source records attributed to Gama, J. B..

4 recordsLinked to original sources

bioRxiv2024

CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization

The endoplasmic reticulum (ER) relies on the microtubule cytoskeleton for distribution and re-modelling of its extended membrane network, but how microtubule-based motors contribute to ER organization remains unclear. Using biochemical and cell-based assays, we identify cerebellar degeneration-related protein 2 (CDR2) and its paralog CDR2-like (CDR2L), onconeural antigens with poorly understood functions, as ER adaptors for cytoplasmic dynein-1 (dynein). We demonstrate that CDR2 is recruited by the integral ER membrane protein kinectin (KTN1) and that double knockout of CDR2 and CDR2L enhances KTN1-dependent ER sheet stacking, reversal of which by exogenous CDR2 requires its dynein-binding CC1 box motif. Exogenous CDR2 expression additionally promotes CC1 box-dependent clustering of ER sheets near centrosomes. CDR2 competes with the eEF1B{beta} subunit of translation elongation factor 1 for binding to KTN1, and eEF1B{beta} knockdown increases endogenous CDR2 levels on ER sheets, inducing their centrosome-proximal clustering. Our study describes a novel molecular pathway that implicates dynein in ER sheet organization and may be involved in the pathogenesis of paraneoplastic cerebellar degeneration.

cell biology↗

bioRxiv2022

The lysosomal adaptor JIP3 activates dynein-dynactin via a short coiled coil

Microtubule motors, like cytoplasmic dynein-1, are tightly regulated to prevent inappropriate activity in cells. Dynein functions as a [~]4 MDa complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognise diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as LIS1 remain unclear. To address this, we determine the cryo-EM structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. We show how JIP3 specifies complex formation despite a shorter coiled coil and lack of identifiable motifs compared to typical adaptors. We find LIS1 directly binds dynactins p150 subunit, closely tethering it to dynein. This interaction is necessary for dyneins cellular and in vitro activity. We also show how dyneins intermediate chain relieves p150s autoinhibition to enable LIS1 binding. Together, our data suggest LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

molecular biology↗

bioRxiv2021

JIP3 regulates bi-directional organelle transport in neurons through its interaction with dynein and kinesin-1

The conserved MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. Whether and how JIP3s interaction with dynein and kinesin-1 contributes to this critical organelle clearance function is unclear. Using purified recombinant human proteins, we show that dynein light intermediate chain (DLIC) binds to the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in a hydrophobic pocket of the RH1 domain, previously shown to abrogate RILPL2 binding to myosin Va, abrogates the binding of JIP3/4 and RILP to DLIC without perturbing the interaction between the JIP3 RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bi-directional organelle transport.

cell biology↗

bioRxiv2018

Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

The kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signalling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, while dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into {micro}m-scale filaments, and we identify the ROD-1 {beta}-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZs capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

cell biology↗