Biology subjects

Muschalik, N.

Publications and source records attributed to Muschalik, N..

4 recordsLinked to original sources

bioRxiv2024

The FAM114A proteins are adaptors for the recycling of Golgi enzymes

The Golgi apparatus contains many resident enzymes that must remain in place whilst their substrates flow through on their journey from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the rims of the Golgi stack to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and at least one COPI adaptor protein, GOLPH3, has been shown to recruit enzymes into vesicles in a specific part of the stack. We have used proximity biotinylation to identify further components of intra-Golgi transport vesicles and found FAM114A2, an uncharacterised cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1 showed that they bind to numerous Golgi resident proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not result in substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Generation of Drosophila mutants lacking FAM114A revealed defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins are COPI vesicle resident proteins that bind to Golgi enzymes and so are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

cell biology↗

bioRxiv2024

Tracking exocytic vesicle movements with MSP-tracker/viewer reveals the spatial control of secretion in epithelial cells.

Understanding how specific secretory cargoes are targeted to distinct domains of the plasma membrane in epithelial cells requires analysing the trafficking of post-Golgi vesicles to their sites of secretion. We used the RUSH (retention using selective hooks) system to synchronously release an apical cargo, Cadherin 99C (Cad99C), and a basolateral cargo, the ECM protein Nidogen, from the endoplasmic reticulum and follow their movements to the plasma membrane. We also developed an interactive vesicle tracking framework, MSP-tracker and viewer, that exploits developments in computer vision and deep learning to determine vesicle trajectories in a noisy environment without the need for extensive training data. MSP-tracker outperformed other tracking software in detecting and tracking post-Golgi vesicles, revealing that Cad99c vesicles predominantly move apically with a mean speed of 1.1{micro}m/sec. This is reduced to 0.85 {micro}m/sec by a dominant slow dynein mutant, demonstrating that dynein transports Cad99C vesicles to the apical cortex. Furthermore, both the dynein mutant and microtubule depolymerisation cause lateral Cad99C secretion. Thus, microtubule organisation plays a central role in targeting apical secretion, suggesting that Drosophila does not have distinct apical versus basolateral vesicle fusion machinery. Nidogen vesicles undergo planar-polarised transport to the leading edge of follicle cells as they migrate over the ECM, whereas most Collagen is secreted at trailing edges. The follicle cells therefore bias secretion of different ECM components to opposite sides of the cell, revealing that the secretory pathway is more spatially organised than previously thought.

cell biology↗

bioRxiv2023

HEATR5B associates with dynein-dynactin and selectively promotes motility of AP1-bound endosomal membranes

The dynein motor complex mediates polarised trafficking of a wide variety of organelles, intracellular vesicles and macromolecules. These functions are dependent on the dynactin complex, which helps recruit cargoes to dyneins tail region and activates motor movement. How dynein and dynactin orchestrate trafficking of diverse cargoes is unclear. Here, we identify HEATR5B, an interactor of the AP1 clathrin adaptor complex, as a novel player in dynein-dynactin function. HEATR5B is one of several proteins recovered in a biochemical screen for proteins whose association with the human dynein tail complex is augmented by dynactin. We show that HEATR5B binds directly to the dynein tail and dynactin and stimulates motility of AP1-associated endosomal membranes in human cells. We also demonstrate that the HEATR5B homologue in Drosophila is an essential gene that promotes dynein-based transport of AP1-bound membranes to the Golgi apparatus. As HEATR5B lacks the coiled-coil architecture typical of dynein adaptors, our data point to a non-canonical process orchestrating motor function on a specific cargo. We additionally show that HEATR5B promotes association of AP1 with endosomal membranes in a dynein-independent manner. Thus, HEATR5B co-ordinates multiple events in AP1-based trafficking.

cell biology↗

bioRxiv2022

Functional unknomics: closing the knowledge gap to accelerate biomedical research

The human genome encodes [~]20,000 proteins, many still uncharacterised. Scientific and social factors have resulted in a focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected. To address this, we have developed an "Unknome database" that ranks proteins based on how little is known about them. We applied RNAi in Drosophila to 260 unknown genes that are conserved between flies and humans. About a quarter are required for viability, and functional screening of the rest revealed hits for fertility, development, locomotion, protein quality control and resilience to stress. CRISPR/Cas9 gene disruption validated a component of Notch signalling and two genes contributing to male fertility. Our work demonstrates the importance of poorly understood genes, provides a resource for future research acceleration, and highlights a need for our awareness of ignorance to be protected from erosion by automated database annotation.

molecular biology↗