Biology subjects

Gifford, L. B.

Publications and source records attributed to Gifford, L. B..

3 recordsLinked to original sources

bioRxiv2025

Heme acts a chloroplast-to-nucleus retrograde signal to regulate intercellular trafficking via plasmodesmata

Intercellular communication via plasmodesmata (PD) is essential for plant growth, development, and defense, yet its regulation remains poorly understood. Chloroplasts communicate information about the environment and the physiological state of the plant cell to the nucleus. It has been proposed that chloroplast-generated signals also modulate the expression of nuclear genes related to PD to regulate the trafficking of photosynthetic products and metabolites along with other molecules that act non-cell-autonomously. In this study, we set out to identify the chloroplast retrograde signals that regulate intercellular trafficking via PD. Using a combination of Arabidopsis thaliana mutants and gene silencing in Nicotiana benthamiana, we found that the metabolites of the tetrapyrrole biosynthetic pathway, most likely heme, can act to modulate PD-mediated intercellular trafficking. We also identified genes that are potentially regulated by the heme signal to modify plasmodesmata function. Together, these findings strengthen the link between chloroplasts and PD in coordinating intercellular communication for optimal plant development and resource allocation.

plant biology↗

bioRxiv2025

TDP-43 directly inhibits RNA accumulation in neurites through modulation of RNA stability

The subcellular localization of hundreds of RNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for the vast majority of these RNAs, the mechanisms that govern their localization are unknown. Here we demonstrate that the ALS-associated RNA binding protein TDP-43 primarily acts to keep RNAs out of neuronal projections. Using subcellular fractionation and single molecule RNA FISH we find that TDP-43 loss results in the increased neurite accumulation of hundreds of RNAs. These RNAs are highly enriched for known TDP-43 binding sites, suggesting that TDP-43 directly binds them to regulate their localization. We then identified precise regions within RNAs that mediate their TDP-43-dependent localization and interaction with TDP-43 using high-throughput functional assays in cells and high-throughput binding assays in vitro. We found that these regions also mediated TDP-43-dependent RNA instability, identifying the mechanism by which TDP-43 regulates RNA localization. ALS-associated mutations in TDP-43 resulted in similar RNA mislocalization phenotypes as did TDP-43 loss in human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of RNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

molecular biology↗

bioRxiv2023

Human Immunodeficiency Virus 1 Capsid Uncoating in the Nucleus Progresses Through Defect Formation in the Capsid Lattice

The HIV-1 core consists of a cone-shaped capsid shell made of [~]250 capsid protein (CA) hexamers and 12 pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is a highly regulated process that is important for productive infection, however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA and visualize capsid trafficking and uncoating in live cells. In addition to direct CA labeling, a fluid phase fluorescent probe is incorporated into the viral core to detect the formation of small defects in the capsid lattice. This double-labeling strategy does not significantly impact HIV-1 infectivity, maturation, nuclear import, or capsid stability. Single virus tracking reveals nuclear import of intact cores defined as complexes containing both the fluid phase marker and robust CA signal. Subsequent uncoating of HIV-1 cores in the nucleus is manifested by a sequential loss of both fluorescent markers. This two-step uncoating - release of the core content marker followed by loss of CA - is observed in different cells, including a macrophage line. Importantly, the lag between the two steps of uncoating ([~]30 min) appears independent of the cell type and is much longer than upon uncoating of cell-free viruses. These data suggest that HIV- 1 uncoating in the nucleus is initiated through a localized defect in the capsid lattice that precedes a global loss of CA. Our results imply that intact HIV-1 cores enter the cell nucleus and uncoat in a stepwise fashion, before integrating into the host genome.

microbiology↗