Biology subjects

Miller, S. I.

Publications and source records attributed to Miller, S. I..

8 recordsLinked to original sources

bioRxiv2026

CFTR dysfunction in the intestinal epithelium is sufficient to promote pathogenic expansion of E. coli and enhanced barrier permeability in cystic fibrosis

Changes in the gut microbiome in cystic fibrosis (CF) are well characterized, yet their causes and downstream effects remain largely unknown. Well-documented alterations include reduced overall complexity (i.e. alpha diversity) of the gut microbiota and increased relative abundance of E. coli, which are associated with greater inflammation and shorter stature in infants. Previous results from our laboratory using a germ-free cystic fibrosis transmembrane conductance regulator (Cftr) mutant mouse model (CF mouse) demonstrated that the observed fecal microbiome dysbiosis is driven by mutated Cftr independent of factors such as diet or antibiotic treatment. We expand on these results in this report by using the defined 8-member community Altered Schaedler Flora (ASF) with and without E. coli, to show that E. coli is pathogenic in the context of the CF gut microbiome, resulting in increased intestinal permeability. We also show that Cftr deletion in intestinal epithelial cells alone, using a Villin-Cre targeted model, is sufficient to raise E. coli abundance in the fecal microbiome, increase intestinal permeability, and amplify the number of TH17 cells in the mesenteric lymph nodes. Together, our results demonstrate that the intestinal epithelium plays a dominant role in fecal microbiome alterations in CF and that the resultant dysbiosis contributes to CF pathogenesis.

microbiology↗

bioRxiv2026

Single-molecule imaging reveals cytoplasmic translation of P granule-enriched mRNAs in C. elegans

Germ granules are condensates in germ plasm, a specialized cytoplasm that segregates to the embryonic germline. In Drosophila, translation of nanos mRNA occurs at the surface of germ granules, suggesting that the granules promote translation. In C. elegans, however, germ (P) granules are not essential for Nanos expression. Using single-molecule imaging in C. elegans embryos, we map the distribution of translating and non-translating molecules of the Nanos homolog nos-2 and two other maternal mRNAs enriched in P granules. In early germline blastomeres, these mRNAs are not translated and distribute between the cytoplasm and P granules. At translation onset, mRNA molecules in the cytoplasm are translated, while most mRNA molecules in the P granules remain non-translating. nos-2 translation requires a rise in the concentration of the RNA-binding protein POS-1, which occurs independently of P granules. Consistent with low translation inside the granules, P granules are depleted of ribosomes and 43S pre-initiation complexes. Our observations suggest that germ granules promote Nanos protein expression by concentrating Nanos mRNA in germline precursors, but do not directly promote translation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/735846v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@7ae911org.highwire.dtl.DTLVardef@15b74fborg.highwire.dtl.DTLVardef@15d1cdforg.highwire.dtl.DTLVardef@129a1a_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisGerm granules are condensates proposed to regulate the translation of mRNAs like Nanos that code for germ cell fate determinants. Using single-molecule imaging in C. elegans embryos, this study shows that P granule scaffolds concentrate mRNAs in germline precursors, but do not control the activity of translational regulators. - P granules concentrate mRNAs but are depleted of ribosomes and 43S pre-initiation complexes - Translation occurs mainly in the cytoplasm where ribosomes are most abundant - nanos translation onset is timed by a rise in POS-1, which counteracts the repressor SPN-4; both enrich in P granules but act independently.

developmental biology↗

bioRxiv2026

Epitope-based labeling for improved live-imaging of endogenous proteins in C. elegans

Visualizing protein expression dynamics with high temporal resolution is essential for understanding how cells acquire specific fates and functions during development, where key decisions can occur within minutes. Conventional direct fluorescent tagging often fails to capture these rapid changes in protein expression due to the relatively slow fluorophore maturation time. Indirect epitope-based labeling strategies offer a promising alternative, yet only a limited number of these systems have been developed and used in the context of multicellular organisms. Here, we evaluate and combine four epitope-based indirect labeling systems for live-imaging of proteins in C. elegans: the SunTag, Frankenbody, MoonTag and AlfaTag systems. Each system uses a fluorescently labeled high-affinity single-chain antibody or nanobody to recognize short peptide epitopes fused to a protein of interest, enabling immediate visualization of newly synthesized proteins. We demonstrate that all four systems specifically label epitope-tagged endogenous proteins and show no detectable cross-reactivity when used in dual-color combinations, enabling simultaneous visualization of distinct proteins within the same embryo. In addition, we show that the SunTag system offers three major advantages over direct labeling: earlier detection of proteins, enhanced sensitivity through signal amplification (as illustrated by CAM-1) and less impact on the function (as demonstrated for ERM-1). Together, this expanded toolkit of epitope-based labeling systems offers many new opportunities for visualizing rapid protein dynamics and for dissecting how their dynamics drive cell fate decisions during development. SUMMARYThe development of epitope-labeling systems has improved live-imaging quality of proteins. Unfortunately, limited systems exist for multicellular organisms to study protein expression in the context of development. Here, we expand the epitope-labeling toolbox for C. elegans by combining SunTag or Frankenbody with MoonTag or AlfaTag. Our data indicates that these systems simultaneously visualize different endogenous proteins without cross-reactivity. Moreover, the SunTag system shows advantages over direct labeling: earlier detection, enhanced sensitivity through signal amplification and less impact on protein function. This expanded epitope-labeling toolbox in C. elegans provides opportunities for accurate visualization of different proteins that drive cell fate decisions. O_FIG O_LINKSMALLFIG WIDTH=155 HEIGHT=200 SRC="FIGDIR/small/703904v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@449fe0org.highwire.dtl.DTLVardef@15c68cforg.highwire.dtl.DTLVardef@1e51ff8org.highwire.dtl.DTLVardef@196114d_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Human genetic variation reveals FCRL3 is a lymphocyte receptor for Yersinia pestis

Yersinia pestis is the gram-negative bacterium responsible for plague, one of the deadliest and most feared diseases in human history. This bacterium is known to infect phagocytic cells, such as dendritic cells and macrophages, but interactions with non-phagocytic cells of the adaptive immune system are frequently overlooked despite the importance they likely hold for human infection. To discover human genetic determinants of Y. pestis infection, we utilized nearly a thousand genetically diverse lymphoblastoid cell lines in a cellular genome-wide association study method called Hi-HOST (High-throughput Human in-vitrO Susceptibility Testing). We identified a nonsynonymous SNP, rs2282284, in Fc receptor like 3 (FCRL3) associated with bacterial invasion of host cells (p=9x10-8). FCRL3 belongs to the immunoglobulin superfamily and is primarily expressed in lymphocytes. rs2282284 is within a tyrosine-based signaling motif, causing an asparagine-to-serine mutation (N721S) in the most common FCRL3 isoform. Overexpression of FCRL3 facilitated attachment and invasion of non-opsonized Y. pestis. Additionally, FCRL3 colocalized with Y. pestis at sites of cellular attachment, suggesting FCRL3 is a receptor for Y. pestis. These properties were variably conserved across the FCRL family, revealing molecular requirements of attachment and invasion, including an Ig-like C2 domain and a SYK interaction motif. Direct binding was confirmed with purified FCRL5 extracellular domain. Following attachment, invasion of Y. pestis was dependent on SYK and decreased with the N721S mutation. Unexpectedly, this same variant is associated with risk of chronic hepatitis C virus infection in BioBank Japan. Thus, Y. pestis hijacks FCRL proteins, possibly taking advantage of an immune receptor to create a lymphocyte niche during infection.

genetics↗

bioRxiv2024

PLK-1 regulates MEX-1 polarization in the C. elegans zygote

The one-cell C. elegans embryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis of C. elegans embryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.

developmental biology↗

bioRxiv2019

SapTrap assembly of C. elegans MosSCI transgene vectors

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans (Schwartz and Jorgensen 2016 Genetics, 202:1277-1288). Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

genetics↗

bioRxiv2018

Identification of Small Molecule Modulators of Diguanylate Cyclase by FRET-based High-Throughput-Screening

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which may make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, may be attractive drug targets to control biofilm formation that is part of chronic infections. In this paper, we present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small molecule modulators of the diguanylate cyclase, DgcA, from Caulobacter crescentus. We identified a set of 7 small molecules that in the low {micro}M range regulate DgcA enzymatic activity. Subsequent structure activity studies on selected scaffolds revealed a remarkable diversity of modulatory behaviors, including slight chemical substitutions that revert the effects from allosteric enzyme inhibition to activation. The compounds identified represent novel chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP associated phenotypes. In sum, our studies underline the importance for detailed mechanism of action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

biochemistry↗

bioRxiv2018

The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

microbiology↗