Biology subjects

Shipman, A.

Publications and source records attributed to Shipman, A..

3 recordsLinked to original sources

bioRxiv2025

ROS inhibits microtubule dynamics and cell growth heterogeneity during Arabidopsis sepal morphogenesis

Developing organs grow to reproducible sizes and shapes yet the growth of their constituent cells can be highly heterogeneous and fluctuating. During wild-type Arabidopsis thaliana sepal development, the fluctuations in cell growth average such that the sepals grow to uniform sizes and shapes. The uniform size and shape of the sepals allow the flower bud to stay closed and protected until the floral organs are mature. In contrast, cell growth averaging is reduced in the ftsh4-5 mutant, and the sepals develop to variable sizes and shapes. FTSH4 encodes a mitochondrial i-AAA protease localized to the mitochondria. Reactive oxygen species (ROS) accumulate in ftsh4-5 mutants, and lowering ROS levels rescues the sepal size and shape variability. Here, we investigate the effects of ROS on cell growth heterogeneity and cortical microtubule dynamics. We find that elevated ROS suppresses cell growth heterogeneity and averaging. We also find that elevated ROS causes cortical microtubules to become more crisscrossed, as well as more stable. The growth of the cells with crisscrossed microtubules changes less in time, which impairs cell growth averaging. However, depolymerizing microtubules is insufficient to restore normal growth fluctuations. Altogether, our results suggest that ROS affects microtubule dynamics and cell growth fluctuations which are necessary for robust morphogenesis.

developmental biology↗

bioRxiv2024

Defects in exosome biogenesis are associated with sensorimotor defects in zebrafish vps4a mutants

Mutations in human VPS4A are associated with neurodevelopmental defects, including motor delays and defective muscle tone. VPS4A encodes a AAA-ATPase required for membrane scission, but how mutations in VPS4A lead to impaired control of motor function is not known. Here we identified a mutation in zebrafish vps4a, T248I, that affects sensorimotor transformation. In biochemical experiments we show the T248I mutation reduces the ATPase activity of Vps4a and disassembly of its substrate, ESCRT filaments, which mediate membrane scission. Consistent with the established role for Vps4a in the endocytic pathway and exosome biogenesis, vps4aT248I mutants have enlarged endosomal compartments in the CNS and decreased numbers of circulating exosomes. Resembling the central form of hypotonia in human VPS4A patients, motor neurons and muscle cells are unaffected in mutant zebrafish as touch sensitivity is intact. Unlike somatosensory function, optomotor responses, vestibulospinal (VS), and acoustic startle reflexes are severely impaired in vps4aT248I mutants, indicating a greater sensitivity of these circuits to the T248I mutation. ERG recordings indicate that visual ability is largely reduced in the mutants, however, in vivo imaging of tone-evoked responses in the inner ear and ascending auditory pathway show comparable activity. Further investigation of central pathways in vps4aT248I mutants revealed that sensory cues failed to fully activate neurons in the VS and medial longitudinal fasciculus (MLF) nuclei that directly innervate motor neurons. Our results suggest that a defect in sensorimotor transformation underlies the profound yet selective effects on motor reflexes resulting from the loss of membrane scission mediated by Vps4a. Significance StatementHere we present a T248I mutation in vps4a, which causes sensorimotor defects in zebrafish larvae. Spanning biochemical to systems level analyses, our study indicates that a reduction in Vps4a enzymatic activity leads to endosomal defects and reduced exosome biogenesis, resulting in pronounced deficits in vision and sensorimotor transformation of auditory and vestibular cues. We suggest that the mechanisms underlying this type of dysfunction in zebrafish may also contribute to the condition seen in human patients with de novo mutations in the human VPS4A orthologue.

neuroscience↗

bioRxiv2018

A screen for targets of the Drosophila pseudokinase Tribbles identifies Neuralized and Mindbomb, ubiquitin ligases that mediate Notch signaling

Drosophila Tribbles (Trbl) is the founding member of a family of pseudokinases with conserved roles in antagonizing cell division, tissue growth and cell differentiation. In humans, three Tribbles isoforms serve as adaptor proteins, binding targets such as Cdc25 phosphatase, Akt kinase or the transcription factor C/EBP to block their activity or direct their proteosomal degradation. Mutations in Tribbles family members are associated with susceptibility to diabetes and cancer, notably Notch-induced tumor growth. Trbl misexpression in the fly wing disk leads to a block in mitosis associated with decreased levels of String/Cdc25 and increased levels of Cyclin B leading to reduced overall wing size and reduced trichome density. We show these Trbl growth-restricting phenotypes can be suppressed by manipulating levels of known Trbl targets, and use this sensitized wing system to screen a collection of growth regulating open reading frames (ORFs) to search for enhancers and suppressors affecting cell and tissue size. By precisely measuring morphometric changes in wing phenotypes using a computer-based tool, we detected synthetic interactions with several E3 ubiquitin ligases, and focused our analysis on the Notch pathway components Neuralized (Neur) and Mindbomb1 (Mib1). In the wing, notum and egg chamber epithelia, Trbl misexpression suppressed Neur and Mib1 activities and stabilized the accumulation of both proteins. To understand these interactions, we used yeast two-hybrid assays to show Trbl physically bound to both Neur and Mib1. Our data are consistent with published reports that mammalian Tribbles3 modulates Notch responses by binding and stabilizing Mindbomb and indicate that a wing misexpression approach is useful to identify novel components in a conserved Tribbles signaling pathway.\n\nAUTHOR SUMMARYTribbles pseudokinases are adaptor molecules, binding diverse targets regulating cell differentiation, growth and proliferation and directing their proteasomal degradation. To search for novel targets of Drosophila Tribbles, we adopted a wing co-misexpression scheme and measured changes in cell/tissue size to identify enhancers and suppressors of the Tribbles phenotype. We show the Notch pathway components Neuralized and Mindbomb1 E3 ligases act as Tribbles suppressors and demonstrate that Tribbles modulates their levels and activites. Recent demonstration that mammalian Tribbles 3 binds the E3 ligase Mindbomb to promote ligand-mediated Notch activation implies a conserved role for Tribbles family members in Notch activation.

developmental biology↗