Biology subjects

Miller, J. C.

Publications and source records attributed to Miller, J. C..

5 recordsLinked to original sources

bioRxiv2025

A method for creating custom 3D-printed molds to facilitate zebrafish imaging studies, including of cardiac development.

Embryo mounting is one of the technical challenges researchers encounter when undertaking an imaging project. Embryos need to be oriented in a reproducible manner such that the tissue of interest is accessible to a microscope objective for the entire imaging period. To overcome this challenge researchers can embed embryos in viscous media or create specialized dishes and casts to hold embryos in a desired orientation during imaging. Here we describe a method for using a cheap stereolithographic (SLA) 3D-printer to manufacture re-usable molds that create agarose wells in which embryos can be mounted for imaging. These agarose wells provide a reliable means for orienting multiple embryos for imaging. This method includes a design framework that can be easily customized for a variety of tissues, organisms and imaging challenges. Using this method we have created molds for imaging cardiac development in zebrafish for both upright and inverted microscopes. By utilizing materials and equipment that are accessible this method allows researchers to easily create molds specific to their mounting needs. SUMMARYHere we describe a method that uses a cheap stereolithographic (SLA) 3D printer to create molds to facilitate the reproducible mounting of zebrafish embryos for imaging studies. Using this method, weve created molds for imaging cardiac morphogenesis in zebrafish embryos.

developmental biology↗

bioRxiv2024

Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

Despite recent progress in the ability to manipulate the genomes of eukaryotic cells1-3, there is still no effective and practical method to precisely integrate large synthetic DNA constructs into desired chromosomal sites using a programmable integrase. Serine integrases can perform the necessary molecular steps4, but only if their natural target site is first installed into the recipient genome by other methods. A more elegant approach would be to directly reprogram the serine integrase itself to target a desired genomic site that is different from the natural recognition site of the integrase5. Here, we describe the development of a Modular Integrase (MINT) platform, a versatile protein-guided genome editing tool that can facilitate site-directed targeted integration (TI) of synthetic DNA into chromosomal sites. Through a combination of structural modeling, directed evolution, and screening in human cells we have reprogrammed the specificity of the serine integrase Bxb1. We demonstrate the therapeutic potential of the MINT platform by retargeting Bxb1 to the human AAVS1 and TRAC loci where wild-type Bxb1 has no detectable activity. By combining the MINT platform with known activity-increasing Bxb1 mutants, we achieved 14% TI at the AAVS1 locus, and by additionally fusing zinc finger DNA binding domains to engineered Bxb1 variants, we achieved 35% TI at the TRAC locus in human K562 cells. To further demonstrate clinical potential, we achieved stable 15% TI of a functional donor construct in human T cells.

synthetic biology↗

bioRxiv2019

Discovery of novel Defense Regulated WD40-repeat proteins DRW1/2 and their roles in plant immunity

Plant heterotrimeric G proteins transduce extracellular signals that activate plant immunity. Plants encode canonical and non-canonical G and G{gamma} subunits, but only a single canonical G{beta} subunit is known. The existence of only one G{beta} subunit limits the number of heterotrimeric G protein combinations able to transduce different signals. It remains unknown whether non-canonical G{beta} subunits exist. Here, we identify two WD40-repeat genes that negatively regulate plant immunity. The proteins encoded by these two genes, DEFENSE REGULATED WD40-REPEAT 1 and 2 (DRW1/2), are structurally similar to AGB1. DRW2 localizes to the plasma membrane and interacts with the canonical G and G{gamma} subunits. Reduced levels of DRW in the drw1 and drw2 single mutants resulted in greater MAPK activation in response to flagellin treatment and conferred increased resistance to the bacterial pathogen Pseudomonas syringae. Furthermore, the drw1 drw2 double-mutant also displayed increased MAPK activation upon flagellin treatment and broad-spectrum resistance against bacterial and fungal pathogen infection. The function of DRW1 and DRW2 is opposite of AGB1, which promotes immune signaling, suggesting that the function of these potential non-canonical G{beta} subunits are not conserved with the canonical G{beta} subunit. Our study identifies additional heterotrimeric G protein components, greatly increasing the number of heterotrimeric G protein complexes that participate in signal transduction.

plant biology↗

bioRxiv2019

Structure-function analysis reveals a DNA polymerization-independent role for mitochondrial DNA polymerase IC in African trypanosomes.

The mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles called kinetoplast DNA (kDNA). Replication of the single nucleoid requires at least three DNA polymerases (POLIB, POLIC, and POLID) each having discrete localization near the kDNA during S phase. POLIB and POLID have roles in minicircle replication while the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNAi-complementation system to dissect the functions of the distinct POLIC domains: the conserved family A DNA polymerase domain (POLA) and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization, active site point mutations in the POLA domain impaired minicircle replication similarly to POLIB and POLID depletions. Complementation with the POLA domain alone abolished POLIC foci formation and partially rescued the RNAi phenotype. Furthermore, we provide evidence of a crucial role for the UCR in cell cycle localization and segregation of kDNA daughter networks. This is the first report of a DNA polymerase that impacts DNA segregation.\n\nSummary statementMitochondrial DNA segregation in African trypanosomes is supported by a dual-functioning DNA polymerase.

microbiology↗

bioRxiv2018

Heterotrimeric G-proteins in unfolded protein response mediate plant growth-defense tradeoffs upstream of steroid and immune signaling

FLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

plant biology↗