Biology subjects

Updegrove, T. B.

Publications and source records attributed to Updegrove, T. B..

5 recordsLinked to original sources

bioRxiv2026

A lipid cue drives the subcellular localization of a self-inserting bacterial transmembrane protein

The correct subcellular localization of proteins is a critical step underlying myriad biological processes, but the cues that drive the specific localization of integral membrane proteins in bacteria remain largely undeciphered. During sporulation in Bacillus subtilis, a rod-shaped outer "mother cell" constructs an internal spherical "forespore" cell that eventually matures into the spore. The integral membrane protein ShfA is made in the mother cell cytosol and localizes to the surface of the forespore. Here, we report that, despite being a multi-pass transmembrane protein, ShfA spontaneously inserts into the lipid bilayer via its N-terminal "YabQ" domain without the apparent need for a pre-localized insertase. ShfA preferentially inserts into cell division septa in multiple bacterial species, indicating that a widely conserved septal cue drives ShfA localization. Structural modeling suggested that the YabQ domain harbors a specific intramembrane groove that can bind the universal lipid carrier undecaprenyl phosphate (UndP), and UndP depletion in vivo disrupted proper ShfA localization. We propose that ShfA localizes to sites of active cell wall synthesis by binding to UndP and/or molecules like lipid I and lipid II that contain UndP and speculate that the function of ShfA is to stabilize these precursors of cell wall biogenesis from the harsh cytosolic nanoenvironment that surrounds the forespore during sporulation.

microbiology↗

bioRxiv2024

Altruistic feeding and cell-cell signaling during bacterial differentiation actively enhance phenotypic heterogeneity

Starvation triggers bacterial spore formation, a committed differentiation program that transforms a vegetative cell into a dormant spore. Cells in a population enter sporulation non-uniformly to secure against the possibility that favorable growth conditions, which puts sporulation-committed cells at a disadvantage, may resume. This heterogeneous behavior is initiated by a passive mechanism: stochastic activation of a master transcriptional regulator. Here, we identify a cell-cell communication pathway that actively promotes phenotypic heterogeneity, wherein Bacillus subtilis cells that start sporulating early utilize a calcineurin-like phosphoesterase to release glycerol, which simultaneously acts as a signaling molecule and a nutrient to delay non-sporulating cells from entering sporulation. This produced a more diverse population that was better poised to exploit a sudden influx of nutrients compared to those generating heterogeneity via stochastic gene expression alone. Although conflict systems are prevalent among microbes, genetically encoded cooperative behavior in unicellular organisms can evidently also boost inclusive fitness.

microbiology↗

bioRxiv2022

Three-dimensional structured illumination microscopy with enhanced axial resolution

We present two distinct, complementary methods for improving axial resolution in three-dimensional structured illumination microscopy (3D SIM) with minimal or no modification to the optical system. First, we show that placing a mirror directly opposite the sample enables 4-beam interference with higher spatial frequency content than 3D SIM illumination, offering near-isotropic imaging with [~]120 nm lateral and 160 nm axial resolution. Second, we develop an improved deep learning method that can be directly applied to 3D SIM data, obviating the need for additional hardware. This procedure results in [~]120 nm isotropic resolution and can be combined with denoising to facilitate volumetric imaging spanning dozens of time points. We demonstrate the potential of these advances by imaging a variety of cellular samples, delineating the nanoscale distribution of vimentin and microtubule filaments, observing the relative positions of caveolar coat proteins and lysosomal markers, and visualizing rich cytoskeletal dynamics within T-cells in the early stages of immune synapse formation.

cell biology↗

bioRxiv2020

Cell-specific cargo delivery using synthetic bacterial spores

SSHELs are synthetic bacterial spore-like particles wherein the spores cell surface is partially reconstituted around 1 {micro}m-diameter silica beads coated with a lipid bilayer. Via a unique cysteine engineered in one of the surface proteins, the surface of SSHELs may be covalently decorated with molecules of interest. Here, we modified SSHELs with an affibody directed against HER2, a cell surface protein overexpressed in some breast and ovarian cancer cells, and loaded them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduced tumor growth with lower toxicity in a mouse tumor xenograft model compared to free drug by specifically binding to HER2-positive cancer cells. We show that SSHELs bound to target cells are taken up and trafficked to acidic compartments, whereupon the cargo is released in a pH-dependent manner. Finally, we demonstrate that SHELLs can clear small tumor lesions in a complex tumor microenvironment in a zebrafish model of brain metastasis. We propose that SSHELs represent a versatile strategy for targeted drug delivery.

synthetic biology↗

bioRxiv2018

Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

Increasing numbers of 3'UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To understand the sequence and structure determinants for the precise processing of the 3' UTR-derived sRNAs, we examined the cleavage of multiple mutant and chimeric derivatives of the 3' UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem-loops 3' to the cleavage site define optimal, correctly-positioned cleavage of MicL and likely other similar sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.

microbiology↗