Biology subjects

Beacham, G. M.

Publications and source records attributed to Beacham, G. M..

7 recordsLinked to original sources

bioRxiv2026

The endothelial scavenger receptor stab2 is required for proper hematopoietic stem and progenitor cell development in the fetal blood stem cell niche

Hematopoietic stem and progenitor cell (HSPC) niches support lifelong production of blood and immune cells. Recently, we identified a gene signature unique to HSPC niche endothelial cells that is highly conserved across species and developmental time and includes the scavenger receptors stab1/2 and mrc1a. Whether these receptors support HSPC development remains unclear. To investigate this, we used chemical inhibition and CRISPR mutagenesis in zebrafish and found that loss of stab2, and to a lesser degree stab1, reduced the number of embryonic runx1(+) HSPCs. Subsequent analyses of an additional HSPC marker (cd41) revealed an imbalance in the HSPC pool in stab2 mutants, with reductions in runx1(+)cd41(+) and runx1(+)cd41(-) sub-populations containing stem cells and erythroid progenitors, respectively, the latter of which was most decreased. Our findings suggest stabilin scavenger receptors support HSPC development in the fetal niche, which could inform clinical strategies for culturing and expanding HSPCs.

developmental biology↗

bioRxiv2025

ASPPs multimerize protein phosphatase 1

Protein Phosphatase 1 (PP1) activity is thought to be spatiotemporally defined by hundreds of different regulatory subunits, but their mechanisms of action are largely unknown. The Ankyrin repeat, SH3-domain, and Proline-rich region containing Proteins (ASPPs) bind and localize PP1 to cell-cell junctions. Here, we show ASPPs bind superstoichiometric amounts of PP1. Missense mutations in the ankyrin repeats of ASPPs, that were previously isolated from a forward genetic screen in Caenorhabditis elegans, reduce the stoichiometry of PP1 binding. Forcing PP1 oligomerization restores mutant ASPP function in vivo. We propose that ASPPs multimerize PP1 to establish a concentrated hub of phosphatase activity at cell-cell junctions. Author SummaryWe have elucidated a new mechanism governing protein phosphatase 1 (PP1) activity. A family of proteins called the ASPPs function to spatially regulate PP1 by recruiting active phosphatase to specific subcellular locations. Critically, we observed that ASPPs promote the formation of higher-order PP1 assemblies - a previously unrecognized regulatory mechanism. We identified specific ASPP mutants in our nematode model organism that disrupt PP1 oligomerization, leading to altered development. However, inducing PP1 clustering was sufficient to rescue these ASPP mutants, underscoring the functional significance of ASPP-mediated PP1 oligomerization. These results provide new insights into the intricate control of cellular signaling pathways mediated by PP1 and may have implications for understanding diseases associated with dysregulated phosphatase activity.

cell biology↗

bioRxiv2024

Dimerization activates the Inversin complex in C. elegans

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologs of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states--an active dimer and an inactive monomer--gates the output of the Inversin complex.

cell biology↗

bioRxiv2024

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O-glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O-glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O-glycosylation in the first larval stage and identify O-glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans, we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/578333v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@68fb6aorg.highwire.dtl.DTLVardef@52166aorg.highwire.dtl.DTLVardef@13dbfaeorg.highwire.dtl.DTLVardef@f352ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

The C. elegans ASPP homolog APE-1 is a junctional protein phosphatase 1 modulator

How serine/threonine phosphatases are spatially and temporally tuned by regulatory subunits is a fundamental question in cell biology. Ankyrin repeat, SH3 domain, proline-rich-region-containing proteins (ASPPs) are protein phosphatase 1 (PP1) binding partners associated with cardiocutaneous diseases. ASPPs localize PP1 to cell-cell junctions, but how ASPPs localize and whether they regulate PP1 activity in vivo is unclear. Through a C. elegans genetic screen, we find that loss of the ASPP homolog, APE-1, suppresses a pathology called jowls, providing us with an in vivo assay for APE-1 activity. Using structure-function analysis, we discover that APE-1s N-terminal half directs the APE-1-PP1 complex to intercellular junctions. Additionally, we isolated mutations in highly conserved residues of APE-1s ankyrin repeats that suppress jowls yet do not preclude PP1 binding, implying ASPPs do more than simply localize PP1. Indeed, in vivo reconstitution of APE-1 suggests the ankyrin repeats modulate phosphatase output, a function we find to be conserved among vertebrate homologs.

cell biology↗

bioRxiv2019

A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in C. elegans. Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.

cell biology↗

bioRxiv2017

NECAPs are negative regulators of the AP2 clathrin adaptor complex

Eukaryotic cells internalize transmembrane receptors via clathrin-mediated endocytosis, but it remains unclear how the machinery underpinning this process is regulated. We recently discovered that membrane-associated muniscin proteins such as FCHo and SGIP initiate endocytosis by converting the AP2 clathrin adaptor complex to an open, active conformation that is then phosphorylated (Hollopeter et al., 2014). Here we report that loss of ncap-1, the sole C. elegans gene encoding an adaptiN Ear-binding Coat-Associated Protein (NECAP), bypasses the requirement for FCHO-1. Biochemical analyses reveal AP2 accumulates in an open, phosphorylated state in ncap-1 mutant worms, suggesting NECAPs promote the closed, inactive conformation of AP2. Consistent with this model, NECAPs preferentially bind open and phosphorylated forms of AP2 in vitro and localize with constitutively open AP2 mutants in vivo. NECAPs do not associate with phosphorylation-defective AP2 mutants, implying that phosphorylation precedes NECAP recruitment. We propose NECAPs function late in endocytosis to inactivate AP2.

cell biology↗