Biology subjects

Wadsworth, P.

Publications and source records attributed to Wadsworth, P..

6 recordsLinked to original sources

bioRxiv2026

A multi-omics map of diabetic nephropathy links c-Jun activation to tubular injury and metabolic stress

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, yet the molecular mechanisms driving tubular injury and fibrosis remain poorly defined. Here, we integrated single-cell multiplexed protein imaging, spatial transcriptomics, single-nucleus and single-cell RNA sequencing and chromatin accessibility profiling to comprehensively characterize human DN pathology. Our multi-modal analysis precisely maps kidney cell types and their spatial distributions, immune-fibrotic interactions, and key transcriptional regulators. We identified eight distinct cellular neighborhoods defining the immune-fibrotic microenvironment and uncovered molecular networks driving tubular injury and fibrosis. JUN (encoding c-Jun) emerged as a central regulator of transcriptional reprogramming during tubular injury and fibrogenic remodeling. In a diabetic mouse model, c-Jun is activated in injured proximal tubules. Using an inducible c-Jun mouse model, we demonstrated that tubular-specific c-Jun activation alone is sufficient to induce tubular injury, chronic inflammation, progressive fibrosis, and systemic metabolic alterations, including impaired glucose homeostasis. We also observed reduced expression of SLC4A4, a bicarbonate transporter essential for proximal tubular function, in injured tubules. Together, our findings establish a spatially resolved framework for understanding DN pathogenesis and identify c-Jun as a key mediator of tubular injury and fibrosis in diabetic kidney disease.

pathology↗

bioRxiv2026

Regulation of midzone microtubule dynamics and abscission in human cells by CAMSAP2 and Kif2a

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

cell biology↗

bioRxiv2023

Human dynein-dynactin is a fast processive motor in living cells

Minus-end directed transport along microtubules in eukaryotes is primarily mediated by cytoplasmic dynein and its cofactor dynactin. Significant advances have been made in recent years characterizing human dynein-dynactin structure and function using in vitro assays, however, there is limited knowledge about the motile properties and functional organization of dynein-dynactin in living human cells. Total internal reflection fluorescence microscopy (TIRFM) of CRISPR-engineered human cells is employed here to visualize fluorescently tagged dynein heavy chain (DHC) and p50 with high spatio-temporal resolution. We find that p50 and DHC exhibit indistinguishable motility properties in their velocities, run lengths, and run times. The dynein-dynactin complexes are fast ([~]1.2 m/s) and run for several microns ([~]2.7 m). Quantification of the fluorescence intensities of motile puncta reveals that dynein-dynactin runs are mediated by at least one DHC dimer while the velocity is consistent with that measured for double dynein (two DHC dimers) complexes in vitro.

cell biology↗

bioRxiv2023

Lateral and longitudinal compaction of PRC1 overlap zones drive stabilization of interzonal microtubules

During anaphase, antiparallel overlapping midzone microtubules elongate and form bundles, contributing to chromosome segregation and the location of contractile ring formation. Midzone microtubules are dynamic in early but not late anaphase; however, the kinetics and mechanisms of stabilization are incompletely understood. Using photoactivation of cells expressing PA-EGF--tubulin we find that immediately after anaphase onset, a single highly dynamic population of midzone microtubules is present; as anaphase progresses, both dynamic and stable populations of midzone microtubules coexist. By midcytokinesis, only static, non-dynamic microtubules are detected. The velocity of microtubule sliding also decreases as anaphase progresses, becoming undetectable by late anaphase. Following depletion of PRC1, midzone microtubules remain highly dynamic in anaphase and fail to form static arrays in telophase despite furrowing. Cells depleted of Kif4a contain elongated zones of PRC1 and fail to form static arrays in telophase. Cells blocked in cytokinesis form short PRC1 overlap zones that do not coalesce laterally; these cells also fail to form static arrays in telophase. Together, our results demonstrate that dynamic turnover and sliding of midzone microtubules is gradually reduced during anaphase and that the final transition to a static array in telophase requires both lateral and longitudinal compaction of PRC1 containing overlap zones.

cell biology↗

bioRxiv2021

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent -tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic - and two mitotic {beta}-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical - and {beta}- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the "brain-eating amoeba" Naegleria fowleri. Using quantitative light microscopy, we find that Naeglerias mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naeglerias spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

cell biology↗

bioRxiv2017

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al. 2014; Ly et al. 2015). Here we show that by using specific intracellular immunolabeling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabeled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, which we term early risers. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

biochemistry↗