Biology subjects

McGurk, A.

Publications and source records attributed to McGurk, A..

4 recordsLinked to original sources

bioRxiv2024

Th1 cells are critical tissue organizers of myeloid-rich perivascular activation niches.

Aggregating immune cells within perivascular niches (PVN) can regulate tissue immunity in infection, autoimmunity and cancer. How cells are assembled at PVNs and the activation signals imparted within remain unclear. Here, we integrate dynamic time-resolved in vivo imaging with a novel spatially-resolved platform for microanatomical interrogation of transcriptome, immune phenotype and inflammatory mediators in skin PVNs. We uncover a complex positive-feedback loop within CXCL10+ PVNs that regulates myeloid and Th1 cell positioning for exchange of critical signals for Th1 activation. Th1 cells spend [~]24h in the PVN, receiving initial peripheral activation signals, before redeploying to the inflamed dermal parenchyma. Niche-enriched, CCR2-dependent myeloid cells were critical for Th1 IFN{gamma}-production. In turn, PVN instructional signals enabled Th1s to orchestrate PVN assembly by CXCR2-dependent intra-tissue myeloid cell aggregation. The results reveal a critical tissue organizing role for Th1s, gained rapidly on tissue entry, that could be exploited to boost regional immunity. HIGHLIGHTSO_LIPerivascular niche (PVN): myeloid hubs in inflamed mouse and healthy human skin C_LIO_LITh1 cells enter, get activated, and leave the PVN within first 24h of tissue entry C_LIO_LIAntigen-specific signals in the PVN promote the tissue organizing functions of Th1s C_LIO_LITh1 cells assemble the PVN via CXCR2-dependent myeloid cell aggregation C_LI

immunology↗

bioRxiv2020

CXCL10+ peripheral activation niches couple preferred sites of Th1 entry with optimal APC encounter

Correct positioning of T cells within infected tissues is critical for T cell activation and pathogen control. Upon tissue entry, effector T cells must efficiently locate antigen presenting cells (APC) for peripheral activation. We reveal that tissue entry and initial peripheral activation of Th1 effector T cells are tightly linked to perivascular positioning of chemokine-expressing APCs. Dermal inflammation induced tissue-wide de novo generation of discrete perivascular CXCL10+ cell clusters, enriched for CD11c+MHC-II+ monocyte-derived dendritic cells. These chemokine clusters were hot spots for both Th1 extravasation and activation in the inflamed skin. CXCR3-dependent Th1 localization to the cluster micro-environment prolonged T-APC interactions and boosted function. Both the frequency and range of these clusters were enhanced via a Th1-intrinsic, IFN{gamma}-dependent positive feedback loop. Thus, the perivascular CXCL10+ clusters act as initial peripheral activation niches, optimizing controlled activation broadly throughout the tissue by coupling Th1 tissue entry with enhanced opportunities for Th1-APC encounter.

immunology↗

bioRxiv2018

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic vs ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44/h45 interface in varying states, that is, engaged, disengaged and in equilibrium. Thirdly, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44/h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

biophysics↗

bioRxiv2018

Structure of the 30S ribosomal decoding complex at ambient temperature

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 [A] resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation.

biophysics↗