Biology subjects

Jackson, P. K.

Publications and source records attributed to Jackson, P. K..

At least 19 recordsLinked to original sources

bioRxiv2025

Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

Pancreatic islet {beta} cells integrate glucose and hormonal cues to control insulin secretion through spatially and temporally organized phosphorylation networks in health and diabetes. Here, using Metabolic STAMP (Synchronized Temporal-Spatial Analysis via Microscopy and Phosphoproteomics), we combine time-resolved phosphoproteomics, imaging, and kinase inhibition in mouse {beta} cells and human islets to map stimulus-specific GPCR signaling pathways. Metabolic STAMP reveals that GLP1-R and FFAR4 engage distinct, compartmentalized kinase programs, including GLP1-R-biased ERK activation, receptor-specific cAMP-PKA domains, and a phospho-ATAT1/HDAC6 node that differentially modulates microtubule acetylation and insulin secretion during GSIS based on stimulation conditions. These GPCR-responsive phospho-signatures and microtubule remodeling patterns are substantially conserved in human islets. Together, our data define an integrated, compartmentalized signaling architecture linking metabolic GPCR inputs, organelle remodeling, and insulin secretion, and provide a {beta}-cell phosphoproteomic resource that connects dynamic signaling nodes to human genetic risk and potential therapeutic targets.

physiology↗

bioRxiv2024

THE FAM53C/DYRK1A axis regulates the G1/S transition of the cell cycle

A growing number of therapies are being developed to target the cell cycle machinery for the treatment of cancer and other human diseases. Consequently, a greater understanding of the factors regulating cell cycle progression becomes essential to help enhance the response to these new therapies. Here, using data from the Cancer Dependency Map, we identified FAM53C as a new regulator of cell cycle progression. We found that FAM53C is critical for this cell cycle transition and that it acts upstream of the CyclinD-CDK4/6-RB axis and of p53 in the regulation of the G1/S transition. By mass spectrometry, biochemical, and cellular assays, we identified and validated DYRK1A as a cell cycle kinase that is inhibited by and directly interacts with FAM53C. Consistent with the role for FAM53C identified in cells in culture, FAM53C knockout human cortical organoids display increased cell cycle arrest and growth defects. Fam53C knockout mice show minor behavioral phenotypes. Because DYRK1A dysregulation contributes to developmental disorders such as Down syndrome as well as tumorigenesis, future strategies aiming at regulating FAM53C activity may benefit a broad range of patients.

cell biology↗

bioRxiv2024

Integrative multiomic approaches reveal ZMAT3 and p21 as conserved hubs in the p53 tumor suppression network

TP53, the most frequently mutated gene in human cancer, encodes a transcriptional activator that induces myriad downstream target genes. Despite the importance of p53 in tumor suppression, the specific p53 target genes important for tumor suppression remain unclear. Recent studies have identified the p53-inducible gene Zmat3 as a critical effector of tumor suppression, but many questions remain regarding its p53-dependence, activity across contexts, and mechanism of tumor suppression alone and in cooperation with other p53-inducible genes. To address these questions, we used Tuba-seqUltra somatic genome editing and tumor barcoding in a mouse lung adenocarcinoma model, combinatorial in vivo CRISPR/Cas9 screens, meta-analyses of gene expression and Cancer Dependency Map data, and integrative RNA-sequencing and shotgun proteomic analyses. We established Zmat3 as a core component of p53-mediated tumor suppression and identified Cdkn1a as the most potent cooperating p53-induced gene in tumor suppression. We discovered that ZMAT3/CDKN1A serve as near-universal effectors of p53-mediated tumor suppression that regulate cell division, migration, and extracellular matrix organization. Accordingly, combined Zmat3-Cdkn1a inactivation dramatically enhanced cell proliferation and migration compared to controls, akin to p53 inactivation. Together, our findings place ZMAT3 and CDKN1A as hubs of a p53-induced gene program that opposes tumorigenesis across various cellular and genetic contexts.

cancer biology↗

bioRxiv2024

Synchronized Temporal-spatial Analysis via Microscopy and Phoshoproteomics (STAMP) of Quiescence

Coordinated cell cycle regulation is essential for homeostasis, with most cells in the body residing in quiescence (G0). Many pathologies arise due to disruptions in tissue-specific G0, yet little is known about the temporal-spatial mechanisms that establish G0 and its signaling hub, primary cilia. Mechanistic insight is limited by asynchronous model systems and failure to connect context-specific, transient mechanisms to function. To address this gap, we developed STAMP (Synchronized Temporal-spatial Analysis via Microscopy and Phospho-proteomics) to track changes in cellular landscape occurring throughout G0 transition and ciliogenesis. For the first time, we synchronized ciliogenesis and G0 transition in two cell models and combined microscopy with phospho-proteomics to order signals for further targeted analyses. We propose that STAMP is broadly applicable for studying temporal-spatial signaling in many biological contexts. The findings revealed through STAMP provide critical insight into healthy cellular functions often disrupted in pathologies, paving the way for targeted therapeutics. TEASERSTAMP of signaling in quiescent cells unravels transient phosphorylations in cellular functions.

cell biology↗

bioRxiv2024

Filopodial Mechanotransduction is regulated by Angiotensin-Converting Enzyme 2 (ACE2) and by SARS-CoV-2 spike protein

Filopodia are dynamic, actin-rich cellular protrusions, increasingly linked to cellular mechanotransduction. However, how dynamic filopodia translate external mechanical cues remains poorly understood. Recent studies show that the SARS-CoV-2 spike (S) protein binds the ACE2 receptor on airway multicilia and that cilia are required for viral infection(1) and sufficient to induce filopodial extension and viral binding. To test if spike protein is sufficient to induce filopodial expansion, we employed live-cell single-particle tracking with quantum dots targeting ACE2, to reveal a robust filopodia extension and virus binding mechanism requiring the enzymatic activity of ACE2. Using time-lapse imaging, we reveal that spike protein binding to filopodia is associated with intracellular actin remodeling, alterations in bulk cell stiffness, and an elevation in intracellular calcium levels linked to actin-rearrangement, filopodia initiation, and persistence. We propose the activation of ACE2 creates an active signaling and mechanosensory environment within adherent cells and airway epithelial cells that allows the remodeling of actin in filopodia to trap virus and potentially organize viral exit from cells. [164 words] One-Sentence SummarySARS-CoV-2 spike protein activates calcium and actin dynamics to enable filopodial extension and virus binding

cell biology↗

bioRxiv2023

A hierarchical pathway for assembly of the distal appendages that organize primary cilia

Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, C3ORF14) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assay revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.

cell biology↗

bioRxiv2023

Myristoylated Neuronal Calcium Sensor-1 captures the ciliary vesicle at distal appendages

The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for thef ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing proper localization to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.

cell biology↗

bioRxiv2022

The IFT81-IFT74 complex enhances GTP hydrolysis to inactivate RabL2 during early steps of intraflagellar transport

Cilia are important organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small Rab-like GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery to regulate the initiation of IFT. We have mapped the interaction with RabL2 to residues 107-195 of CEP19, purified the RabL2-CEP19 complex to show that CEP19 is not a GTPase activator protein for RabL2. In contrast, a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis in RabL2 by 20-fold. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 is mapped to a 70 amino acid long coiled-coil region of IFT81/74. We present structural models for minimal IFT81/74-RabL2 complexes and demonstrate that the Chlamydomonas IFT81/74 complex enhances GTP hydrolysis of human RabL2 suggesting an ancient evolutionarily conserved function. Our results provide a mechanistic understanding of RabL2 function in the initiation step of IFT and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.

biochemistry↗

bioRxiv2022

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration

In aging, skeletal muscle strength and regenerative capacity declines due, in part, to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here we capitalize on mass-cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blocking of CD47 alternative polyadenylation or antibody blockade of CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

cell biology↗

bioRxiv2021

The Mettl3 epitranscriptomic writer amplifies p53 stress responses

The p53 transcription factor, encoded by the most frequently mutated gene in human cancer, plays a critical role in tissue homeostasis in response to stress signals. The mechanisms through which p53 promotes downstream tumor suppressive gene expression programs remain, however, only superficially understood. Here, we used tandem affinity purification and mass spectrometry to reveal new components of the p53 response. This approach uncovered Mettl3, a component of the m6A RNA methyltransferase complex (MTC), as a p53-interacting protein. Analysis of Mettl3- deficient cells revealed that Mettl3 promotes p53 protein stabilization and target gene expression in response to DNA damage. Mettl3 acts in part by competing with the p53 negative regulator, Mdm2, for binding to the p53 transactivation domains to promote methyltransferase-independent stabilization of p53. In addition, Mettl3 relies on its catalytic activity to augment p53 responses, with p53 recruiting Mettl3 to p53 target genes to co-transcriptionally direct m6A modification of p53 pathway transcripts to enhance their expression. Mettl3 also promotes p53 activity downstream of oncogenic signals in vivo, in both allograft and autochthonous lung adenocarcinoma models, suggesting cooperative action of p53 and Mettl3 in tumor suppression. Accordingly, we found in diverse human cancers that mutations in MTC components perturb expression of p53 target genes and that MTC mutations are mutually exclusive with TP53 mutations, suggesting that the MTC enhances the p53 transcriptional program in human cancer. Together, these studies reveal a fundamental role for Mettl3 in amplifying p53 signaling through protein stabilization and epitranscriptome regulation.

cancer biology↗

bioRxiv2021

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

Oncogenic KRAS mutations occur in approximately 30% of lung adenocarcinoma. Despite several decades of effort, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we used multiplexed somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing. Through a series of CRISPR/Cas9 screens in autochthonous lung tumors, we identified HRAS and NRAS as key suppressors of KRASG12D-driven tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, RAS paralogs interact with oncogenic KRAS, suppress KRAS-KRAS interactions, and reduce downstream ERK signaling. HRAS mutations identified in KRAS-driven human tumors partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRAS- and BRAF-driven lung cancer models confirmed that RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner in vivo and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven lung cancer.

cancer biology↗

bioRxiv2020

Divergent molecular signatures of regeneration and fibrosis during wound repair

Regeneration is the "holy grail" of tissue repair, but skin injury typically yields fibrotic, non-functional scars. Developing pro-regenerative therapies requires rigorous understanding of the molecular progression from injury to fibrosis or regeneration. Here, we report the divergent molecular events driving skin wound cells toward either scarring or regenerative fates. We profile scarring versus YAP inhibition-induced wound regeneration at the transcriptional (single-cell RNA-sequencing), protein (timsTOF proteomics), and tissue (extracellular matrix ultrastructural analysis) levels. Using cell surface barcoding, we integrate these data to reveal fibrotic and regenerative "molecular trajectories" of healing. We show that disrupting YAP mechanical signaling yields regenerative repair orchestrated by fibroblasts with activated Trps1 and Wnt signaling. Our findings serve as a multimodal map of wound regeneration and could have therapeutic implications for pathologic fibroses.

cell biology↗

bioRxiv2020

Ethacridine inhibits SARS-CoV-2 by inactivating viral particles in cellular models

SARS-CoV-2 is the coronavirus that causes the respiratory disease COVID-19, which is now the third-leading cause of death in the United States. The FDA has recently approved remdesivir, an inhibitor of SARS-CoV-2 replication, to treat COVID-19, though recent data from the WHO shows little to no benefit with use of this anti-viral agent. Here we report the discovery of ethacridine, a safe antiseptic use in humans, as a potent drug for use against SARS-CoV-2 (EC50 ~ 0.08 M). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescent assay. Interestingly, the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Indeed, ethacridine is effective in various cell types, including primary human nasal epithelial cells. Taken together, these data identify a promising, potent, and new use of the old drug possessing a distinct mode of action for inhibiting SARS-CoV-2.

microbiology↗

bioRxiv2020

Discovery of ciliary G protein-coupled receptors regulating pancreatic islet insulin and glucagon secretion

Multiple G protein coupled receptors (GPCRs) are expressed in pancreatic islet cells but the majority have unknown functions. We observe specific GPCRs localized to primary cilia, a prominent signaling organelle, in pancreatic - and {beta}-cells. Loss of cilia disrupts {beta}-cell endocrine function, but the molecular drivers are unknown. Using functional expression, we identified multiple GPCRs localized to cilia in mouse and human islet - and {beta}-cells, including FFAR4, PTGER4, DRD5, ADRB2, KISS1R, and P2RY14. Free fatty acid receptor 4 (FFAR4) and prostaglandin E receptor 4 (PTGER4) agonists stimulate ciliary cAMP signaling and promote glucagon and insulin secretion by - and {beta}-cell lines, and by mouse and human islets. Transport of GPCRs to primary cilia requires TULP3, whose knockdown in primary human and mouse islets depleted ciliary FFAR4 and PTGER4, and impaired regulated glucagon or insulin secretion, without affecting ciliary structure. Our findings provide index evidence that regulated hormone secretion by islet - and {beta}-cells is regulated by ciliary GPCRs providing new targets for diabetes.

physiology↗

bioRxiv2020

A CRISPR-based genome-wide screen for adipogenesis reveals new insights into mitotic expansion and lipogenesis.

In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and by upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional genomics screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. The pooled screening strategy utilized FACS to isolate populations based on lipid content by gating for fluorescence intensity of the lipophilic, green fluorescent BODIPY dye. Additionally, the approach categorized if genes functioned during mitotic expansion or lipogenesis. Cellular mechanisms regulating the rate of protein translation and protein stability were found critical for adipogenesis and lipogenesis. These protein-directed mechanisms were further supported by proteomic analyses, which demonstrated that essential changes in protein abundance driving 3T3-L1 adipogenesis were not driven by transcription. We exemplify this theme by showing that the hypusination pathway, a conserved regulator of translation initiation, is critical to translate adipogenic inducers of mitotic expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity to regulate lipogenesis.

molecular biology↗

bioRxiv2020

Multiple domains in ARHGAP36 regulate PKA degradation and Gli activation

ARHGAP36 is a Rho GTPase-activating protein (GAP) family member that contributes to spinal cord development and tumorigenesis. This multidomain protein is composed of splicing-dependent N-terminal sequences, the GAP-like region, and a unique C-terminal domain, and an N-terminal arginine-rich region has been shown to suppress protein kinase A (PKA) and activate Gli transcription factors. To understand how these structural elements act in concert, we have mapped the ARHGAP36 structure-activity landscape with domain- and amino-acid-level resolution. ARHGAP36-mediated Gli activation can be repressed by N-terminal sequences that regulate subcellular ARHGAP36 localization and PKA targeting. The GAP-like and C-terminal domains counteract this autoinhibitory mechanism and promote ARHGAP36 trafficking to the plasma membrane and primary cilium, respectively. The GAP-like domain may also conditionally suppress the arginine-rich region, and it modulates ARHGAP36 binding to the prolyl oligopeptidase-like protein PREPL and the E3 ubiquitin ligase PRAJA2. These domain-dependent activities provide a potential means for tissue-specific ARHGAP36 functions.

cell biology↗

bioRxiv2020

Combined Proteomic and Genetic Interaction Mapping Reveals New RAS Effector Pathways and Susceptibilities

Activating mutations in RAS GTPases drive one fifth of cancers, but poor understanding of many RAS effectors and regulators, and of the roles of their different paralogs, continues to impede drug development. We developed a multi-stage discovery and screening process to understand RAS function and identify RAS-related susceptibilities in lung adenocarcinoma. Using affinity purification mass spectrometry (AP/MS), we generated a protein-protein interaction map of the RAS pathway containing thousands of interactions. From this network we constructed a CRISPR dual knockout library targeting 119 RAS-related genes that we screened for genetic interactions (GIs). We found important new effectors of RAS-driven cellular functions, RADIL and the GEF RIN1, and over 250 synthetic lethal GIs, including a potent KRAS-dependent interaction between RAP1GDS1 and RHOA. Many GIs link specific paralogs within and between gene families. These findings illustrate the power of the multiomic approach to identify synthetic lethal combinations for hitherto undruggable cancers. STATEMENT OF SIGNIFICANCEWe present a thorough survey of protein-protein and genetic interactions in the Ras pathway. These interactions suggested new discoveries that we validate here, and demonstrate important new paralog specificities and redundancies. By comparing synthetic lethal interactions across KRAS-dependent and -independent tumors, we identify new combination therapy targets against Ras-driven cancers.

cancer biology↗

bioRxiv2020

Ciliation of muscle stem cells is critical to maintain regenerative capacity and is lost during aging

During aging, the regenerative capacity of muscle stem cells (MuSCs) decreases, diminishing the ability of muscle to repair following injury. We performed a small molecule library screen and discovered that the proliferation and expansion of aged MuSCs is regulated by signal transduction pathways organized by the primary cilium, a cellular protrusion that serves as a sensitive sensory organelle. Abolishing MuSC cilia in vivo severely impaired injury-induced muscle regeneration. In aged muscle, a cell intrinsic defect in MuSC ciliation leading to impaired Hedgehog signaling was associated with the decrease in regenerative capacity. This deficit could be overcome by exogenous activation of Hedgehog signaling which promoted MuSC expansion, both in vitro and in vivo. Delivery of the small molecule Smoothened agonist (SAG) to muscles of aged mice restored regenerative capacity leading to increased strength post-injury. These findings provide fresh insights into the signaling dysfunction in aging and identify the ciliary Hedgehog signaling pathway as a potential therapeutic target to counter the loss of muscle regenerative capacity which accompanies aging.

cell biology↗