Biology subjects

Moore, J. K.

Publications and source records attributed to Moore, J. K..

10 recordsLinked to original sources

bioRxiv2026

Tubulin C-terminal tails are pH sensors that regulate microtubule function

Changes in intracellular pH are critical for maintaining homeostasis, mediating signaling pathways, and enabling cellular responses to stress, injury, and disease. There is increasing evidence that clusters of acidic residues, primarily glutamates, are both highly prevalent and conserved in disordered regions of proteins and can play an important role in cellular pH response. Tubulin C-terminal tails (CTTs) are glutamate rich regions which protrude from the microtubule surface. These tails are a primary site of for both post-translational modifications and binding of microtubule-associated proteins. Motivated by these observations, we measured the pH response of tubulin CTTs using NMR spectroscopy, circular dichroism, and computational simulations. We find that glutamate residues in CTTs taken from organisms across eukaryotes exhibit a robust upshift in their pKa values, that the sequential context of glutamate residues creates hot spots for protonation, and that hydrogen bonding between side chains stabilizes interactions that alter the conformation of the CTT. To determine whether the CTT pH response plays a potentially important role in microtubule interactions, we measured the pH dependence of the binding of the yeast kinesin-5, Cin8, to microtubules. We find that Cin8 binding is modulated by pH in a CTT-dependent manner. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish that pH can regulate interactions at the microtubule surface. Significance StatementVariation in cellular pH is important for cell function in changing environmental conditions or developmental states. Here we probe protonation of the glutamate-rich C-terminal tails of tubulin, revealing the existence of and mechanism driving the anomalously high pH response and subsequent regulation of microtubule binding. Our results demonstrate that acidic clusters are important mediators of cellular pH response and establish pH-based regulation of interactions at the microtubule surface.

biophysics↗

bioRxiv2026

Role of α-tubulin helix 11' in heterodimer conformation and microtubule dynamics

Tubulin heterodimers transition from a curved conformation in solution to straight conformation when assembled into the microtubule lattice. Many proteins and small molecules alter microtubule dynamics by binding and stabilizing the curved or straight conformations; however, we have a poor understanding of the regions of the tubulin heterodimer that contribute to this transition and whether these conformations represent sources of phenotypic variation in human disease and across species. We previously identified a role for -tubulin helix 11 (H11) in the curved-to-straight transition. Using computational simulations and tubulin mutants in budding yeast, we show that H11 variants associated with human disease destabilize the curved conformation of the heterodimer and disrupt -tubulin function in cells. Comparing -tubulin sequences across eukaryotes demonstrates strong conservation of H11 with the exception of several -tubulin isotypes expressed during mitosis in the amoebae Naegleria fowleri and Naegleria gruberi. Introducing H11 sequence variants from Naegleria in budding yeast -tubulin increases heterodimer exchange at microtubule plus ends and destabilizes mitotic spindles. We provide evidence that interactions between -tubulin H11 and {beta}-tubulin H8 stabilize the curved conformation of tubulin heterodimers, and that the equilibrium between curved and straight conformations is an ancient feature of tubulin evolution.

cell biology↗

bioRxiv2024

Selective regulation of kinesin-5 function by β-tubulin carboxy-terminal tails

The tubulin code hypothesis predicts that carboxy-terminal tails of tubulins create programs for selective regulation of microtubule-binding proteins, including kinesin motors. However, the molecular mechanisms that determine selective regulation and their relevance in cells are poorly understood. In this work, we report selective regulation of budding yeast kinesin-5 motors, Cin8 and Kip1, by the {beta}-tubulin tail. Cin8, but not Kip1, requires the {beta}-tubulin tail for its recruitment to the mitotic spindle. This creates a balance of both kinesin-5 motors in the spindle, and efficient mitotic progression. We identify a negatively charged patch of acidic amino acids in the {beta}-tubulin tail that mediates the interaction with Cin8 in vivo. Using in vitro reconstitution with genetically modified yeast tubulin, we demonstrate that the charged patch in the {beta}-tubulin tail increases Cin8 plus-end directed velocity and processivity. Finally, we determine that the positively charged amino-terminal extension from the Cin8 motor domain coordinates interactions with the {beta}-tubulin tail. This work provides the first demonstration of a molecular mechanism underlying selective regulation of closely related kinesin motors by tubulin tails, and how this regulation promotes proper function of the mitotic spindle.

cell biology↗

bioRxiv2023

Quantifying yeast microtubules and spindles using the Toolkit for Automated Microtubule Tracking (TAMiT)

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that is solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work, and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

cell biology↗

bioRxiv2022

The RNA-binding protein EIF4A3 promotes axon development by direct control of the cytoskeleton

The exon junction complex (EJC), nucleated by EIF4A3, is indispensable for mRNA fate and function throughout eukaryotes. Unexpectedly, we discover that EIF4A3 directly controls microtubules independent of RNA, and this is critical for neural wiring. While neuronal survival in the developing mouse cerebral cortex depends upon an intact EJC, axonal tract formation requires only Eif4a3. Using human cortical organoids, we demonstrate that EIF4A3 disease mutations also impair neuronal maturation, highlighting conserved functions relevant for neurodevelopmental pathology. Employing biochemistry and molecular modeling we discover that EIF4A3 directly binds to microtubules, mutually exclusive of the RNA-binding complex. In growing neurons, EIF4A3 is essential for microtubule dynamics, and sufficient to promote microtubule polymerization and stability in vitro. Together, our data show that tubulin-bound EIF4A3 orchestrates microtubule dynamics, underlying key events of neuronal development. This reveals a new mechanism by which neurons re-utilize core gene expression machinery to rapidly and directly control the cytoskeleton. HighlightsO_LIThe Exon Junction Complex controls neuronal survival but only EIF4A3 directs axonal growth C_LIO_LIEIF4A3 controls axonal tract formation in vivo. C_LIO_LIHuman EIF4A3 deficient iPSC-derived cortical organoids recapitulate neuronal defects. C_LIO_LIEIF4A3 directly binds to microtubules to control their growth and stability in neurons. C_LI

neuroscience↗

bioRxiv2022

Asymmetric requirement for α-tubulin over β-tubulin

How cells regulate the supply of - and {beta}-tubulin monomers to meet the demand for {beta}- heterodimers while avoiding consequences of monomer imbalance is not understood. We investigate the role of gene copy number in tubulin regulation and how shifting the expression of - or {beta}-tubulin genes impacts tubulin proteostasis and microtubule function. We find that - tubulin gene copy number is important for maintaining an excess -tubulin protein compared to {beta}-tubulin protein and preventing accumulation of super-stoichiometric {beta}-tubulin. Super- stoichiometric {beta}-tubulin is toxic to cells, leading to loss of microtubules, formation of non- microtubule assemblies of tubulin, and disrupted cell proliferation. In contrast, decreased {beta}- tubulin or increased -tubulin has minor effects. We provide evidence that cells rapidly equilibrate the concentration of -tubulin protein during shifts in -tubulin isotype expression to maintain a ratio in excess of {beta}-tubulin. We propose an asymmetric relationship between - and {beta}-tubulins, where -tubulins are maintained in excess to supply {beta}-heterodimers and limit the accumulation of {beta}-tubulin monomers.

cell biology↗

bioRxiv2021

Tubulinopathy mutations in TUBA1A that disrupt neuronal morphogenesis and migration override XMAP215/Stu2 regulation of microtubule dynamics

Heterozygous, missense mutations in - or {beta}-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutations impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.

cell biology↗

bioRxiv2020

Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone

How cells regulate microtubule crosslinking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control spindle elongation rate. Using budding-yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved, basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces faster elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.

cell biology↗

bioRxiv2020

Tuba1a is uniquely important for axon guidance through midline commissural structures

Developing neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of /{beta}-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of -tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a -tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

developmental biology↗

bioRxiv2019

Systemic Lymphoid Architecture Response Assessment (SYLARAS): An approach to multi-organ, discovery-based immunophenotyping implicates a role for CD45R/B220+ CD8T cells in glioblastoma immunology

Accurately profiling systemic immune responses to cancer initiation and progression is necessary for understanding tumor surveillance and, ultimately, improving therapy. Here, we describe the SYLARAS software tool (SYstemic Lymphoid Architecture Response ASsessment) and a data set collected with SYLARAS that describes the frequencies of immune cells in primary and secondary lymphoid organs and in the tumor microenvironment of mice engrafted with a standard syngeneic glioblastoma (GBM) model. The data resource involves profiles of 5 lymphoid tissues in 48 mice and shows that GBM causes wide-spread changes in the local and systemic immune architecture. We perform in-depth analysis of one significant tumor-induced change: depletion of a specialized subset of CD45R/B220+ CD8+ T cells from the circulation and their accumulation in the tumor mass. Immunoprofiling of tissue microarrays demonstrates the presence of similar cells in human GBM.

immunology↗