An official website of the United States government
Abstract The orientation of the mitotic spindle at metaphase determines the placement of the daughter cells. Spindle orientation in animals typically relies on an evolutionarily conserved biological machine comprised of at least four proteins – called Pins, Gαi, Mud, and Dynein in flies – that exerts a pulling force on astral microtubules and reels the spindle into alignment. The canonical model for spindle orientation holds that the direction of pulling is determined by asymmetric placement of this machinery at the cell cortex. In most cell types, this placement is thought to be mediated by Pins, and a substantial body of literature is therefore devoted to identifying polarized cues that govern localized cortical enrichment of Pins. In this study we revisit the canonical model and find that it is incomplete. Spindle orientation in theDrosophilafollicular epithelium and embryonic ectoderm requires not only Pins localization but also direct interaction between Pins and the multifunctional protein Discs large. This requirement can be over‐ridden by interaction with another Pins interacting protein, Inscuteable.
more »
« less
Drosophila Adducin facilitates phase separation and function of a conserved spindle orientation complex
Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. InDrosophilaneural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). Here we identify Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (HtsHEAD) to bind a short Mud coiled-coil domain (MudCC) that is adjacent to the Pins-binding domain (MudPBD). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation.In vitroanalyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, we find that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via MudCColigomerization) and protein disorder (via an intrinsically disordered region in Hts; HtsIDR) in phase separation of the Hts/Mud/Pins complex. Our study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output.
more »
« less
- Award ID(s):
- 2205405
- PAR ID:
- 10489476
- Publisher / Repository:
- Frontiers
- Date Published:
- Journal Name:
- Frontiers in Cell and Developmental Biology
- Volume:
- 11
- ISSN:
- 2296-634X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
ABSTRACT The orientation of the mitotic spindle determines the direction of cell division, and therefore contributes to tissue shape and cell fate. Interaction between the multifunctional scaffolding protein Discs large (Dlg) and the canonical spindle orienting factor GPSM2 (called Pins in Drosophila and LGN in vertebrates) has been established in bilaterian models, but its function remains unclear. We used a phylogenetic approach to test whether the interaction is obligate in animals, and in particular whether Pins/LGN/GPSM2 evolved in multicellular organisms as a Dlg-binding protein. We show that Dlg diverged in C. elegans and the syncytial sponge Opsacas minuta and propose that this divergence may correspond with differences in spindle orientation requirements between these organisms and the canonical pathways described in bilaterians. We also demonstrate that Pins/LGN/GPSM2 is present in basal animals, but the established Dlg-interaction site cannot be found in either Placozoa or Porifera. Our results suggest that the interaction between Pins/LGN/GPSM2 and Dlg appeared in Cnidaria, and we therefore speculate that it may have evolved to promote accurate division orientation in the nervous system. This work reveals the evolutionary history of the Pins/LGN/GPSM2-Dlg interaction and suggests new possibilities for its importance in spindle orientation during epithelial and neural tissue development.more » « less
-
Edelstein-Keshet, Leah (Ed.)The search-and-capture model of spindle assembly has been a guiding principle for understanding prometaphase for decades. The computational model presented allows one to address two questions: how rapidly the microtubule–kinetochore connections are made, and how accurate these connections are. In most previous numerical simulations, the model geometry was drastically simplified. Using the CellDynaMo computational platform, we previously introduced a geometrically and mechanically realistic 3D model of the prometaphase mitotic spindle, and used it to evaluate thermal noise and microtubule kinetics effects on the capture of a single chromosome. Here, we systematically investigate how geometry and mechanics affect a spindle assembly’s speed and accuracy, including nuanced distinctions between merotelic, mero-amphitelic, and mero-syntelic chromosomes. We find that softening of the centromere spring improves accuracy for short chromosome arms, but accuracy disappears for long chromosome arms. Initial proximity of chromosomes to one spindle pole makes assembly accuracy worse, while initial chromosome orientation matters less. Chromokinesins, added onto flexible chromosome arms, allow modeling of the polar ejection force, improving a spindle assembly’s accuracy for a single chromosome. However, spindle space crowding by multiple chromosomes worsens assembly accuracy. Our simulations suggest that the complex microtubule network of the early spindle is key to rapid and accurate assembly.more » « less
-
Barrier-to-autointegration (BAF) is a DNA binding protein that crosslinks chromatin to allow mitotic nuclear envelope (NE) assembly. The Lap2b-Emerin-Man1(LEM)-domain protein LEMD2 and ESCRTII/III hybrid protein CHMP7 close NE holes surrounding spindle microtubules (MTs). BAF binds LEM-domain family proteins, which repairs NE ruptures in interphase, but whether BAF-LEM binding participates in NE hole closure around spindle MTs is not known. Here, we take advantage of the stereotypical event of NE formation in fertilized C. elegans oocytes to show that BAF-LEM binding and LEM-2LEMD2-CHMP-7 have distinct roles in NE closure around spindle MTs. LEM-2/EMR-1emerin function redundantly with BAF-1 in NE closure. Compromising BAF-LEM binding revealed an additional role for EMR-1emerin in maintenance of the NE permeability barrier. In the absence of BAF-LEM binding, LEM-2-CHMP-7 are required for NE assembly and embryo survival. The winged helix domain of LEM-2 recruits CHMP-7 to the NE in C. elegans and a LEM-2-independent nucleoplasmic pool of CHMP-7 also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis.more » « less
-
ABSTRACT The body plan of the human parasite Toxoplasma gondii has a well-defined polarity. The minus ends of the 22 cortical microtubules are anchored to the apical polar ring, which is a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end and is crucial for cytokinesis. How this apical–basal polarity is initiated is unknown. Here, we have examined the development of the apical polar ring and the basal complex using expansion microscopy. We found that substructures in the apical polar ring have different sensitivities to perturbations. In addition, apical–basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the nascent daughter framework grows towards the centrioles, the apical and basal arcs co-develop ahead of the microtubule array. Finally, two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of individual proteins has a modest impact on the lytic cycle. However, the loss of both proteins results in abnormalities in the microtubule array and in highly reduced plaquing and invasion efficiency.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fcell.2023.1220529
