Biology subjects

Manning, G.

Publications and source records attributed to Manning, G..

5 recordsLinked to original sources

bioRxiv2025

Changes in nuclear and actin mechanics from G1 to G2 affect nuclear integrity

The structural integrity of the nucleus is dependent on nuclear mechanical elements of chromatin and lamins to resist antagonistic actin cytoskeleton forces. Imbalance results in nuclear blebbing, rupture, and cellular dysfunction found in many human diseases. We used Fluorescent Ubiquitin Cell Cycle Indicator (FUCCI) cells to determine how cell cycle changes affect the nucleus and actin force balance. While nuclear blebs are present equally throughout interphase, nuclear blebs form predominantly in G1 and then persist into G2 due to increased actin-based nuclear confinement and focal adhesion density in G1 vs. G2 cells. Upon artificial confinement, G2 nuclei ruptured more than G1 nuclei. Single nucleus micromanipulation force measurements confirmed that G1 nuclei are stronger than G2 nuclei in both the chromatin-based and lamin-based nuclear stiffness regimes. Decreased nuclear stiffness can be explained by loss of peripheral H3K9me3 from G1 to G2, recapitulated by H3K9me3 inhibition via Chaetocin. Cell cycle-based changes in nuclear and actin mechanics impact nuclear integrity and shape.

cell biology↗

bioRxiv2025

Lamin B loss in nuclear blebs is rupture dependent while increased DNA damage is rupture independent

The nucleus houses genetic information and functions separate from the rest of the cell. Loss of nuclear shape results in nuclear ruptures. Nuclear blebs are deformations identified by decreased DNA density, while lamin B levels vary drastically. To determine if decreased lamin B levels are due to nuclear rupture, we used immunofluorescence to measure levels of lamin B and emerin, a nuclear envelope protein that enriches to sites of nuclear rupture. We observed that cell types that exhibit decreased levels of lamin B also show an enrichment of emerin in nuclear blebs. Oppositely, in other cell types, nuclear blebs display maintained levels of lamin B1 and showed no emerin enrichment. To determine how nuclear rupture affects DNA damage, we time lapse imaged nuclear rupture dynamics then fixed the same cells to conduct immunofluorescence of {gamma}H2AX and emerin. We find that DNA damage levels are higher in blebbed nuclei independent of nuclear rupture. Thus, we confirm that lamin B1 loss in nuclear blebs is due to nuclear rupture and blebbed nuclei have increased DNA damage that is independent of rupture. Summary statement (180-200 characters): We measured lamin B and DNA damage in blebbed nuclei to determine the effect of nuclear rupture. We find that nuclear rupture causes loss of lamin B in nuclear blebs but that increased DNA damage in blebbed nuclei is independent of rupture.

cell biology↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2024

Unconventional binding of Calmodulin to CHK2 kinase inhibits catalytic activity

Calmodulin (CaM) serves an essential role in eukaryotic cells as a Ca2+ sensor. Ca2+ binding leads to conformation changes in CaM that enable engagement of a repertoire of enzymes and the regulation of their catalytic activities. Classically, Ca2+-CaM binds to an inhibitory pseudosubstrate sequence C-terminal to the kinase domain in members of the Ca2+-CaM dependent protein kinase (CAMK) family, and relieves inhibition to promote catalytic activity. Here, we report an unexpected mechanism by which CaM can bind CHK2 kinase to inhibit its kinase activity. Using biochemical, biophysical, structural mass spectrometry, and cellular approaches, we identify a direct interaction of Ca2+-CaM with the CHK2 kinase domain that suppresses CHK2 catalytic activity in vitro and is crucial for cell proliferation in human cells following DNA damage. Our findings add direct suppression of kinase activity to the repertoire of CaMs functions, complementing the paradigmatic mechanism of promoting kinase activity through autoinhibitory domain sequestration.

biochemistry↗

bioRxiv2022

An RNA sequence that reprograms ribosomes to bypass a 50 nucleotide coding gap is encoded by a mobile element whose sequence conservation illuminates its bypass mechanisms

BackgroundA remarkable sequence in phage T4 causes ribosomes to skip over a 50 nucleotide insert within a topoisomerase subunit gene, and resume correct synthesis of the protein at a high efficiency. Its mechanism has been extensively studied but it remained an isolated phenomenon whose origin and full function are still a mystery. ResultsWe have found dozens of homologous cases in genomic and metagenomic sequences, all part of a mobile DNA element that repeatedly inserts in topoisomerase genes of Myoviridae phages. These have substantial sequence diversity, with selective conservation that specify the elaborate set of mechanisms found experimentally to underlie this extreme case of translational recoding. These sequences provide new variations on these mechanisms, and introduce additional features that may also be important for bypassing. These include a series of RNA secondary structures, a conserved stop codon or rare hungry codon at the start of the bypass, a Shine-Dalgarno sequence flanked by AU-rich sequence, and residues in the nascent peptide that prime the ribosome for bypassing. ConclusionsThese data provide an evolutionary foundation for the experimentally derived mechanisms, highlight several new features of the sequence, and provide substantial new variations on the bypass theme that will allow further experimental exploration of biologically meaningful variants.

biochemistry↗