Biology subjects

Patterson, G. H.

Publications and source records attributed to Patterson, G. H..

4 recordsLinked to original sources

bioRxiv2024

Using Photoswitching FRET to Define the Interaction Boundaries between the Rab1b GTPase and Secretory Cargo

FRET is a powerful tool to simultaneously establish and localize interactions between fluorescently tagged proteins with high spatial resolution. Rainey K.H. and Patterson G.H. introduced Photoswitching FRET (psFRET) using the Dronpa Photoswitching fluorescent protein. We present a straightforward detailed method, and a powerful software tool that allows adaptation of psFRET to diverse experimental setups. Image stacks, recording the decay of the Dronpa donor, serve as input to the software utility that includes effective preprocessing options preceding the calculation FRET efficiency at the single pixel level. We applied psFRET to generate interaction maps analyzing diverse interactions between cargo proteins, the GTPase Rab1b, and GRASP65 during ER to Golgi trafficking. Cargo-Rab1b interactions were restricted to the transit period from ER to Golgi. These data lend support to a mechanism whereby cargo sensing may regulate the level of downstream effectors recruitment to secretory membranes by Rab1.

cell biology↗

bioRxiv2021

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant increase in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within molecular condensates.

biophysics↗

bioRxiv2019

Uncoating of COPII from ER exit site membranes precedes cargo accumulation and membrane fission

COPII and COPI are considered to be analogous sets of vesicle coat protein heterocomplexes. Coupled to cargo selection, they mediate the formation of membrane vesicles translocating in opposite directions to differ rent destinations within the secretory pathway. Here, live cell and electron microscopy provided evidence for a different localization and mode of function of the COPII coat during protein export from the endoplasmic reticulum (ER). Pharmaceutical and genetic perturbations of ER-Golgi transport were used to demonstrate that COPII is recruited to membranes defining the boundary of ER-ER Exit Sites (ERES) where it facilitates selective cargo concentration. Uncoating of COPII membranes precedes cargo accumulation and fission of Golgi-bound carriers. Moreover, we report what may be direct transfer of cargo to the Golgi apparatus from Golgi-associated BFA sensitive ERESs. Finally, in ldlF cells the stably expressed functional {varepsilon}-COPI-EYFP labeled both ERESs and anterograde carriers. These findings change our understanding of the role of coat proteins in ER to Golgi transport.

cell biology↗

bioRxiv2017

Single-shot super-resolution total internal reflection fluorescence microscopy

We demonstrate a simple method for combining instant structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF), doubling the spatial resolution of TIRF (down to 115 +/-13 nm) and enabling imaging frame rates up to 100 Hz over hundreds of time points. We apply instant TIRF-SIM to multiple live samples, achieving rapid, high contrast super-resolution imaging in close proximity to the coverslip surface.

bioengineering↗