Biology subjects

Johnson, C. P.

Publications and source records attributed to Johnson, C. P..

9 recordsLinked to original sources

bioRxiv2025

Macrophages induce stromal differentiation and endothelialization in iPSC-derived kidney organoids.

Human induced pluripotent stem cell (iPSC) - derived kidney organoids are powerful models of kidney development and disease but lack mature vasculature. Here, we show that co-culture with human monocyte-derived macrophages (MDM) induces robust endothelial and stromal differentiation in iPSC-derived kidney organoids. MDM promoted the formation of lumenized capillary networks, an effect reproduced by iPSC-derived macrophages and the THP-1 monocyte line. In contrast, organoids without MDM displayed only transient endothelial differentiation that coincided with transient VEGFA upregulation and that regressed during maturation. MDM counteracted this transient phase by releasing soluble Neuropilin-1 (sNRP1), which sequestered VEGFA and stabilized late endothelial development, evidenced by sustained CD31 promoter activity. Transcriptomic profiling revealed that MDM redirected mesodermal fate suppressing lateral plate and cardiac mesoderm while promoting paraxial and intermediate mesoderm and the generation of FoxD1 stromal progenitors via CXCL5 (ENA-78) secretion. These stromal cells supported late endothelial maturation and enhanced overall organoid differentiation. Our findings uncover a macrophage-driven mechanism coupling stromal and vascular development, providing a strategy to achieve functional vascularization in kidney organoids and improving their physiological relevance for research applications.

cell biology↗

bioRxiv2025

Glucocorticoid and Mineralocorticoid Receptors Jointly Promote Vascular Development in Kidney Organoids

To examine the co-development of vasculature and renal epithelial tissue, we employed a human pluripotent stem cell-derived kidney organoid system. We found that cooperative signaling through the glucocorticoid receptor and mineralocorticoid receptor via hydrocortisone enabled rich endothelial cell differentiation and vessel formation. Bulk RNA sequencing analysis revealed that hydrocortisone perturbs an angiogenic transcriptional program early in development and promotes instead a pro-endothelial survival transcriptional program, with upregulation of angiopoietin 1 at both the mRNA and protein level. Additionally, we saw that hydrocortisone does not seem to significantly affect gene expression of canonical nephrogenic genes compared to our controls, suggesting its effect is largely restricted to endothelial cell differentiation. Our results show that kidney organoids offer a unique platform to study developmental signals that drive endothelial cell differentiation and vessel formation.

developmental biology↗

bioRxiv2025

SGLT2 Inhibition Ameliorates Age-Dependent Renovascular Rarefaction

IntroductionAging is associated with progressive loss of renal function and vascular structure, with and without chronic kidney disease. However, the mechanisms driving renal vascular aging and potential therapeutic interventions remain poorly understood. MethodsTo model this state-of-affairs, we used African turquoise killifish (Nothobranchius furzeri), a naturally short-lived vertebrate. We then inhibited the sodium-glucose co-transporter 2 using dapagliflozin (SGLT2i) to test a potential therapeutic intervention. Histological, immunofluorescent, and 3D vascular imaging were used to evaluate glomerular, tubular, vascular and functional changes. Single-nuclei transcriptomic profiling was performed on whole kidneys to identify age- and treatment-associated molecular signatures. ResultsAged killifish kidneys exhibited hallmark features of renal aging, including glomerulosclerosis, tubular fibrosis, and vascular rarefaction. Functional changes included increased proteinuria and altered tubular transporter function. Transcriptomic profiling revealed a metabolic shift from oxidative phosphorylation to glycolysis and upregulation of pro-inflammatory pathways. Aged vasculature also displayed a marked reduction in tight junctions and cell-cell contacts. SGLT2i attenuated age-related vascular rarefaction, preserved functional capillary networks, reduced albuminuria, restored a youthful transcriptional profile and enhanced intercellular signaling. However, killifish lifespan was not extended. ConclusionThis study establishes the killifish as a translational model for investigating renal vascular aging. We show that SGLT2i preserves renal microvascular structure and function, reduces proteinuria, and reprograms the aged transcriptome. These results support a vascular-protective role of SGLT2i in mitigating age-related renal deterioration. Translational StatementThis study establishes the African turquoise killifish as a model for investigating renal and vascular aging. We found that SGLT2 inhibition preserves microvascular integrity and reduces proteinuria. These results mirror established benefits observed in mammalian models and patients with chronic kidney disease, reinforcing the kidney-protective role of SGLT2 inhibitors. However, the killifish offers a unique opportunity for rapid, translational aging research. By using a naturally short-lived vertebrate with mammalian-like renal aging, our model enables a rapid, preclinical, assessment of vascular outcomes and identifies microvascular preservation as a potential mechanistic target for renoprotection.

systems biology↗

bioRxiv2025

Ferlin C2A-C2B linkers are alternatively spliced, intrinsically disordered, and interact with negatively charged membranes

Ferlins are vesicle trafficking proteins composed of folded C2 domains conjugated by linkers which are largely disordered. Although a role for the for the C2 domains as calcium sensors has been established it remains unclear whether the linkers function beyond acting as passive spacers. We examined the C2A-C2B linker of vertebrate ferlins and found both putative AP2 and SH3 binding short linear motifs (SLiMs) as well as membrane binding sequences for members of the protein family. Specifically for otoferlin we identified an arginine-rich region proximal to a AP2 binding dileucine motif which interacts with negatively charged lipid membranes. Further, the linker region dominated the liposome binding properties of a larger C2A-C2B two-C2 domain segment of otoferlin, suggesting a dominant role in mediating the membrane binding property of the N-terminus. We also found that alternative splicing of the otoferlin C2A-C2B linker adds and additional membrane binding segment and alters the affinity and kinetics of membrane binding. By contrast alternative splicing of the dysferlin linker is not predicted to alter membrane binding but rather alters the number of predicted short linear motifs (SLiMs). In addition we found the otoferlin linker-membrane interaction was sensitive to ionic strength, and simulations suggest positively charged residues including an arginine-rich region mediates binding. We conclude that the C2A-C2B linker of vertebrate ferlins encode both SLiMs which recruit endocytic proteins as well as membrane binding regions that would place the endocytic binding motif proximal to the membrane surface to facilitate endocytosis and synaptic vesicle resupply.

molecular biology↗

bioRxiv2024

TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

Target of Rapamycin (TOR) complexes and G-protein-coupled receptors (GPCRs) are crucial signaling hubs that coordinate adaptive responses to environmental inputs. While GPCR-mediated regulation of TOR has been extensively studied, little is known about TOR-mediated regulation of GPCRs. Here, we establish TOR as a regulator of GPCR signaling via its control of receptor endocytosis in the yeast mating system. By pairing fluorescence microscopy with yeast genetic approaches, we identify the machinery that bridges TOR nutrient sensing to GPCR internalization. Furthermore, we find that autophagic machinery is employed during mating to deliver active receptor to the vacuole (lysosome), suppressing the mating pathway. These results demonstrate that TOR regulates the localization and signaling of the yeast mating GPCR in both ligand-dependent and -independent contexts. These pathways are highly conserved, suggesting that TOR-regulation of GPCRs may be a broadly conserved mechanism for integrating competing signals involving metabolic state and external communications.

cell biology↗

bioRxiv2023

The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery

The septin cytoskeleton plays a key role in the morphogenesis of the yeast mating projection, forming structures at the base of the projection. The yeast mating response uses the G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. Desensitization of the G, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. We hypothesized that Gpa1 would utilize known septin regulators to control septin organization. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the endocytic adaptor proteins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive G. We hypothesized that hyperactive G might increase the rate of endocytosis of a pheromone-responsive cargo, thereby altering where septins are localized. Mathematical modeling predicted that changes in endocytosis could explain the septin organizations we find in WT and mutant cells. Our results show that Gpa1-induced disorganization of septins requires clathrin-mediated endocytosis. Both the GPCR and the G are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deleting the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the G delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.

cell biology↗

bioRxiv2021

Dynamic Phosphorylation of RGS Provides Spatial Regulation of G-alpha And Promotes Completion of Cytokinesis

Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.

cell biology↗

bioRxiv2021

Calcium Sensitive Allostery Regulates the PI(4,5)P2 Binding Site of the Dysferlin C2A Domain

C2 domains are the second-most abundant calcium binding module in the proteome. Activity of the muscular dystrophy associated protein dysferlin is dependent on the C2A domain at the N-terminus of the protein, which couples calcium and PI(4,5)P2 binding through an unknown mechanism. Using solution state nuclear magnetic resonance spectroscopy we confirm the phosphoinositide binding site for the domain and find that calcium binding attenuates millisecond to microsecond motions at both in the calcium binding loops and the concave face of the C2A, including a portion of the phosphoinositide binding site. Our results support a model whereby increasing calcium concentrations shift the phosphoinositide binding pocket of C2A into a binding-competent state, allowing for calcium dependent membrane targeting. This model contrasts with the canonical mechanism for C2 domain-phosphoinositide interaction and provides a basis for how pathogenic mutations in the C2A domain result in loss of function and disease.

biophysics↗

bioRxiv2018

Neuropathy and neural plasticity in the subcutaneous white adipose depot.

The difficulty in obtaining as well as maintaining weight loss, together with the loss of metabolic control in conditions like diabetes and cardiovascular disease, may represent pathological situations of inadequate neural communication between the brain and peripheral organs and tissues. Innervation of adipose tissues by peripheral nerves provides a means of communication between the master metabolic regulator in the brain (chiefly the hypothalamus), and energy-expending and energy-storing cells in the body (primarily adipocytes). Although chemical and surgical denervation studies have clearly demonstrated how crucial adipose tissue neural innervation is for maintaining proper metabolic health, we have uncovered that adipose tissue becomes neuropathic in various conditions of metabolic dysregulation. Here, utilizing both human and mouse adipose tissues, we present evidence of adipose tissue neuropathy, or loss of innervation, under pathophysiological conditions such as obesity, diabetes, and aging, all of which are concomitant with insult to the adipose organ and metabolic dysfunction. Neuropathy is indicated by loss of nerve fiber protein expression, reduction in synaptic markers, and less neurotrophic factor expression in adipose tissue. Aging-related adipose neuropathy particularly results in loss of innervation around the tissue vasculature. These findings underscore that peripheral neuropathy is not restricted to classic tissues like the skin of distal extremities, and that loss of innervation to adipose may trigger or exacerbate metabolic diseases. In addition, we have demonstrated stimulation of adipose tissue neural plasticity with exercise, cold exposure or neurotrophic factor treatment, which may ameliorate adipose neuropathy and be a potential therapeutic option to re-innervate adipose and restore metabolic health.

neuroscience↗