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Spindle formation in male meiosis relies on the canonical centrosome system, which is distinct from acentrosomal oocyte meiosis, but its specific regulatory mechanisms remain unknown. Herein, we report that DYNLRB2 (Dynein light chain roadblock-type-2) is a male meiosis-upregulated dynein light chain that is indispensable for spindle formation in meiosis I. In Dynlrb2 KO mouse testes, meiosis progression is arrested in metaphase I due to the formation of multipolar spindles with fragmented pericentriolar material (PCM). DYNLRB2 inhibits PCM fragmentation through two distinct pathways; suppressing premature centriole disengagement and targeting NuMA (nuclear mitotic apparatus) to spindle poles. The ubiquitously expressed mitotic counterpart, DYNLRB1, has similar roles in mitotic cells and maintains spindle bipolarity by targeting NuMA and suppressing centriole overduplication. Our work demonstrates that two distinct dynein complexes containing DYNLRB1 or DYNLRB2 are separately used in mitotic and meiotic spindle formations, respectively, and that both have NuMA as a common target. Male meiosis relies on canonical centrosomes for spindle formation, but how this differs from acentrosomal oocyte meiosis is unclear. Here they show that spindle formation in sperm relies on DYNLRB2, similar to the activity of DYNLRB1 in mitotic cells.

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis.

The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair; however, this ability is not engaged in all neurons and injury locations. Here, we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron (MN) axons in Drosophila larvae. The dileucine zipper kinase (DLK; also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila ) is a key regulator of diverse responses to axonal injury. In three different populations of MNs, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spared even a small part of a synaptic terminal were insufficient to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.

The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair; however, this ability is not engaged in all neurons and injury locations. Here, we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron (MN) axons in Drosophila larvae. The dileucine zipper kinase (DLK; also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila ) is a key regulator of diverse responses to axonal injury. In three different populations of MNs, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spared even a small part of a synaptic terminal were insufficient to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.

Cytoplasmic dynein-1 (dynein) is the primary retrograde-directed microtubule motor in most eukaryotes. To be active, dynein must bind to the dynactin complex and a cargo-specific adaptor to form the . There are nearly 20 adaptors that, despite having low sequence identity, all contain two discrete domains that mediate binding to the same regions of dynein and dynactin. Additionally, all adaptors seem to generate active transport complexes with grossly similar structures. Despite these similarities, active transport complexes formed with different adaptors show differences in their velocity, run length, and microtubule binding affinity. The molecular features in adaptors that underlie the differences in activity is unknown. To address this question, we first generated a library of synthetic adaptors by deleting or systematically swapping characterized dynein and dynactin binding domains for four endogenous, model adaptors, NINL, BicD2, KASH5, and Hook3. We then used binding assays and TIRF-based motility assays to assess each synthetic adaptors' ability to bind and activate dynein and dynactin. First, we found that the adaptors' coiled-coil domains, which bind dynactin and the tail domain of dynein, are necessary and sufficient for dynein activation. Second, we found that all endogenous adaptors could be modified to yield a synthetic adaptor that formed more motile active transport complexes, which suggests that there is no selective pressure for adaptors to maximize dynein motility. Indeed, our data suggest that some endogenous adaptor sequences may have evolved to generate active transport complexes that are only moderately motile. Finally, we found that one synthetic adaptor was hyperactive and generated active transport complexes that moved faster, farther, and more frequently than all other endogenous and synthetic adaptors. By performing structure-function analyses with the hyperactive adaptor, we discovered that increased random coil at key positions in an adaptor sequence increases the likelihood that dynein-dynactin-adaptor complexes that assemble will be motile. Our work supports a model where increased adaptor flexibility facilitates a type of kinetic proofreading that specifically destabilizes improperly assembled and inactive dynein-dynactin-adaptor complexes. These results provide insight into how differences in adaptor sequences could contribute to differential dynein regulation.

Cytoplasmic dynein-1 (dynein) is a microtubule-associated, minus end-directed motor that traffics hundreds of different cargos. Dynein must discriminate between cargos and traffic them at the appropriate time from the correct cellular region. How dynein's trafficking activity is regulated in time or cellular space remains poorly understood. Here, we identify CCSer2 as the first known protein to gate dynein activity in the spatial dimension. CCSer2 promotes the migration of developing zebrafish primordium cells and of cultured human cells by facilitating the trafficking of cargos that are acted on by cortically localized dynein. CCSer2 inhibits the interaction between dynein and its regulator Ndel1 exclusively at the cell periphery, resulting in localized dynein activation. Our findings suggest that the spatial specificity of dynein is achieved by the localization of proteins that disinhibit Ndel1. We propose that CCSer2 defines a broader class of proteins that activate dynein in distinct microenvironments via Ndel1 inhibition.

Dynein harnesses ATP hydrolysis to move cargo on microtubules in multiple biological contexts. Dynein meets a unique challenge in meiosis by moving chromosomes tethered to the nuclear envelope to facilitate homolog pairing essential for gametogenesis. Though processive dynein motility requires binding to an activating adaptor, the identity of the activating adaptor required for dynein to move meiotic chromosomes is unknown. We show that the meiosis-specific nuclear-envelope protein KASH5 is a dynein activating adaptor: KASH5 directly binds dynein using a mechanism conserved among activating adaptors and converts dynein into a processive motor. We map the dynein-binding surface of KASH5, identifying mutations that abrogate dynein binding in vitro and disrupt recruitment of the dynein machinery to the nuclear envelope in cultured cells and mouse spermatocytes in vivo. Our study identifies KASH5 as the first transmembrane dynein activating adaptor and provides molecular insights into how it activates dynein during meiosis. Competing Interest Statement The authors have declared no competing interest.

The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair, however this ability is not engaged in all neurons and injury locations. Here we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron axons in Drosophila larvae. The dileucine zipper kinase DLK, (also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila), is a key regulator of diverse responses to axonal injury. In three different populations of motoneurons, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spare even a small part of a synaptic terminal fail to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.Competing Interest StatementThe authors have declared no competing interest.

Background: Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. Methods: We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. Results: Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor–cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA’s binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. Conclusions: This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA–VSIG axis to enhance anti-tumor responses.