The internal Cdc20 binding site in BubR1 facilitates both spindle assembly checkpoint signalling and silencing

Improperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyze the binding of two checkpoint proteins, Mad2 and BubR1, to Cdc20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of Cdc20 kinetochore localization in the SAC, we delineate the molecular details of its interaction with kinetochores. We find that BubR1 recruits the bulk of Cdc20 to kinetochores through its internal Cdc20 binding domain (IC20BD). We show that preventing Cdc20 kinetochore localization by removal of the IC20BD has a limited effect on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second Cdc20 binding site in BubR1 in promoting both efficient SAC signaling and SAC silencing

The coding and long noncoding single-cell atlas of the developing human fetal striatum.

Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment

Regulation of mitotic progression by the spindle assembly checkpoint

Equal segregation of sister chromatids during mitosis requires that pairs of kinetochores establish proper attachment to microtubules emanating from opposite poles of the mitotic spindle. The spindle assembly checkpoint (SAC) protects against errors in segregation by delaying sister separation in response to improper kinetochore–microtubule interactions, and certain checkpoint proteins help to establish proper attachments. Anaphase entry is inhibited by the checkpoint through assembly of the mitotic checkpoint complex (MCC) composed of the 2 checkpoint proteins, Mad2 and BubR1, bound to Cdc20. The outer kinetochore acts as a catalyst for MCC production through the recruitment and proper positioning of checkpoint proteins and recently there has been remarkable progress in understanding how this is achieved. Here, we highlight recent advances in our understanding of kinetochore–checkpoint protein interactions and inhibition of the anaphase promoting complex by the MCC

Distinct domains in Bub1 localize RZZ and BubR1 to kinetochores to regulate the checkpoint

The spindle assembly checkpoint (SAC) ensures proper chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Checkpoint signalling requires the kinetochore localization of the Mad1–Mad2 complex that in more complex eukaryotes depends on the Rod–Zwilch–ZW10 (RZZ) complex. The kinetochore protein Zwint has been proposed to be the kinetochore receptor for RZZ, but here we show that Bub1 and not Zwint is required for RZZ recruitment. We find that the middle region of Bub1 encompassing a domain essential for SAC signalling contributes to RZZ localization. In addition, we show that a distinct region in Bub1 mediates kinetochore localization of BubR1 through direct binding, but surprisingly removal of this region increases checkpoint strength. Our work thus uncovers how Bub1 coordinates checkpoint signalling by distinct domains for RZZ and BubR1 recruitment and suggests that Bub1 localizes antagonistic checkpoint activities

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation

Brain Regional Identity and Cell Type Specificity Landscape of Human Cortical Organoid Models.

Peer reviewed: TrueIn vitro models of corticogenesis from pluripotent stem cells (PSCs) have greatly improved our understanding of human brain development and disease. Among these, 3D cortical organoid systems are able to recapitulate some aspects of in vivo cytoarchitecture of the developing cortex. Here, we tested three cortical organoid protocols for brain regional identity, cell type specificity and neuronal maturation. Overall, all protocols gave rise to organoids that displayed a time-dependent expression of neuronal maturation genes such as those involved in the establishment of synapses and neuronal function. Comparatively, guided differentiation methods without WNT activation generated the highest degree of cortical regional identity, whereas default conditions produced the broadest range of cell types such as neurons, astrocytes and hematopoietic-lineage-derived microglia cells. These results suggest that cortical organoid models produce diverse outcomes of brain regional identity and cell type specificity and emphasize the importance of selecting the correct model for the right application