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Oriented cell division is fundamental to development and tissue organization, requiring precise control of both spindle positioning and orientation. While cortical pulling forces mediated by dynein motor proteins are well-established drivers of spindle dynamics, the contribution of microtubule polymerization-based pushing forces remains unclear. We developed a generalizable computational biophysical model that integrates both pulling and pushing mechanisms to investigate spindle behavior across diverse cell types and geometries. This model successfully recapitulates experimental observations in three well-studied models:Drosophilafollicular epithelial cells,Drosophilaneuroblasts, and the earlyC. elegansembryo. Systematic analysis reveals that while pulling forces are the primary drivers of directed spindle orientation, pushing forces play crucial supporting roles by preventing spindle stalling and promoting alignment dynamics, particularly at high initial misalignment angles. We further applied our model to irregularly shaped zebrafish endothelial cells, which present unique challenges due to their non-spherical morphology and dynamic shape changes during mitosis. Our results demonstrate that asymmetric cortical force generator distributions, potentially localized at cell-cell junctions, can account for the observed off-center spindle positioning in these cells. This work provides a unified framework for understanding how the interplay between cell geometry, molecular polarity cues, and competing physical forces determines spindle dynamics, offering new insights into both canonical and non-canonical division orientations across cell types. 

Abstract Mud/NuMA/LIN-5 (in flies, vertebrates, and worms) is an evolutionarily conserved protein that regulates the shape and orientation of the mitotic spindle. In vertebrate cells, these functions depend on a C-terminal region called the NuMA-Tip, which (i) mediates interaction with the conserved partner protein LGN (called Pins in flies), (ii) contains a highly conserved subsequence called the NLM, and (ii) binds directly to microtubule ends. Although Mud plays a vital role in Drosophila mitosis, less is known about its structure, particularly at the C-terminus. Through sequence analysis and functional studies, we identify the Mud-Tip region and show that it is encoded by 3 consecutive exons. These exons are spliced out of several Mud isoforms, creating functionally distinct “Tipless” variants. We find that Tipless isoforms are specifically expressed in male and female gametes, where they localize to the nuclear envelope. Although Mud is known to be essential for female fertility, we demonstrate that this function does not require an intact Tip region. We also find that Mud antagonizes the localization of Lamin, a nucleoskeletal protein, in the testis, and uncover an unexpected role for Tipless Mud in promoting male fertility. Our work reveals that while the Mud-Tip is important for Mud function at mitosis, alternative splicing ensures this region is absent from Mud isoforms that perform a moonlighting role during meiosis. 

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule “tubulin code,” which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament. 

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.