Biology subjects

Griffis, E.

Publications and source records attributed to Griffis, E..

5 recordsLinked to original sources

bioRxiv2023

Glycolytic Metabolon Assembly on Mitochondria via Hexokinase O-GlcNAcylation Promotes Metabolic Efficiency

Glucose, the primary cellular energy source, is metabolized through glycolysis initiated by the rate-limiting enzyme Hexokinase (HK). In energy-demanding tissues like the brain, HK1 is the dominant isoform, primarily localized on mitochondria, crucial for efficient glycolysis-oxidative phosphorylation coupling and optimal energy generation. This study unveils a unique mechanism regulating HK1 activity, glycolysis, and the dynamics of mitochondrial coupling, mediated by the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes reversible O-GlcNAcylation, a post-translational modification, influenced by glucose flux. Elevated OGT activity induces dynamic O-GlcNAcylation of HK1s regulatory domain, subsequently promoting the assembly of the glycolytic metabolon on the outer mitochondrial membrane. This modification enhances HK1s mitochondrial association, orchestrating glycolytic and mitochondrial ATP production. Mutations in HK1s O-GlcNAcylation site reduce ATP generation, affecting synaptic functions in neurons. The study uncovers a novel pathway that bridges neuronal metabolism and mitochondrial function via OGT and the formation of the glycolytic metabolon, offering new prospects for tackling metabolic and neurological disorders.

cell biology↗

bioRxiv2023

Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.

Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport because anterograde vesicles are marked with a different Rab than retrograde vesicles. To explore this proposal, we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab, Ypt51. The Sec2GEF-GFP-CUE construct was found to localize to bright puncta predominantly near sites of polarized growth and this localization was strongly dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. The puncta appeared to consist of clusters of 80 nm vesicles and although the puncta are largely static, FRAP analysis suggests that traffic into and out of these clusters continues. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth, where they tethered and fused with the plasma membrane despite the misdirection of Sec4 and its effectors. In total, the results suggest that while Rabs play a critical role in regulating vesicular transport, cells are remarkably tolerant of Rab misdirection.

cell biology↗

bioRxiv2023

The lung employs an intrinsic surfactant-mediated inflammatory response for viral defense

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles surfactant deficient RDS. Using a novel multi-cell type, human induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that inflammatory cytokine/chemokine production and interferon (IFN) responses are dynamically regulated autonomously within the lung following SARS-CoV-2 infection, an intrinsic defense mechanism mediated by surfactant proteins (SP). Single cell RNA sequencing revealed broad infectability of most lung cell types through canonical (ACE2) and non-canonical (endocytotic) viral entry routes. SARS-CoV-2 triggers rapid apoptosis, impairing viral dissemination. In the absence of surfactant protein B (SP-B), resistance to infection was impaired and cytokine/chemokine production and IFN responses were modulated. Exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion restored resistance to SARS-CoV-2 and improved viability.

immunology↗

bioRxiv2020

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.

immunology↗

bioRxiv2017

FAM83G/PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP

Our previous studies of PAWS1 (Protein Associated With SMAD1) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1 knockout cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration.\n\nSummary statementPAWS1/FAM83G controls cell migration by influencing the organisation of F-actin and focal adhesions and the distribution of the actin stress fibre network through its association with CD2AP.

cell biology↗