Molecular Mechanism of Conserved Vps45 in Nascent Lipid Droplet Motility
Project Description
Background: Lipid droplets (LDs) are essential for cellular lipid homeostasis. They are evolutionarily conserved organelles that originate from the endoplasmic reticulum (ER). Yeast ER-vacuole contact sites (nucleus-vacuole junctions, NVJs) act as specialized hubs that expand during metabolic stress to facilitate LD biogenesis. While actin‑based transport later distributes LDs throughout the cell, the mechanisms that mobilize LDs immediately after they mature at the ER are still poorly understood.
Preliminary data and Research aim: Vps45, a highly conserved protein that regulates membrane fusion, functions as a tethering factor within the endosomal system. VPS45 deficiency leads to an extremely rare, life-threatening autosomal recessive disorder, also known as congenital neutropenia-myelofibrosis-nephromegaly syndrome. Surprisingly, our preliminary data reveal that yeast cells lacking Vps45, but not the endosomal protein Vps35, exhibit strong LD accumulation between the ER and vacuole, specifically at the periphery of NVJs – the LD biogenesis site. Although LD size and number remain unchanged, LD mobility is significantly reduced. Notably, Vps45 partially colocalizes with LDs, and we observe dynamic “kiss-and-run” events between Vps45 and LDs. We thus hypothesize that Vps45 is required for mobilizing nascent LDs from the ER, potentially functioning together with tether or motor proteins to enable subsequent LD trafficking to other cellular locations. To test this hypothesis, we will mechanistic dissection of the Vps45’s role in licensing LD motility within the yeast ER network.
Research methodology: Our approach integrates advanced microscopy, biochemistry, and genetics to elucidate the Vps45's role in transitioning LDs from a static to a motile state. We will assess potential functional relationships between Vps45 and known LD tether protein Mdm1, and the motor protein Myo2 – by determining their localization relative to Erg6-marked LDs in wt and vps45Δ yeast cells using advanced fluorescence microscopy. Mistargeting of any of these proteins in vps45Δ cells will prompt co-immunoprecipitation (Co-IP) studies, followed by confirmation of interactions through mutant analysis. In parallel, We will identify novel Vps45 interactors using affinity purification-mass spectrometry with GFP- or TurboID-tagged Vps45, validate candidates biochemically, and determine their functional roles in LD mobility.
Preliminary data and Research aim: Vps45, a highly conserved protein that regulates membrane fusion, functions as a tethering factor within the endosomal system. VPS45 deficiency leads to an extremely rare, life-threatening autosomal recessive disorder, also known as congenital neutropenia-myelofibrosis-nephromegaly syndrome. Surprisingly, our preliminary data reveal that yeast cells lacking Vps45, but not the endosomal protein Vps35, exhibit strong LD accumulation between the ER and vacuole, specifically at the periphery of NVJs – the LD biogenesis site. Although LD size and number remain unchanged, LD mobility is significantly reduced. Notably, Vps45 partially colocalizes with LDs, and we observe dynamic “kiss-and-run” events between Vps45 and LDs. We thus hypothesize that Vps45 is required for mobilizing nascent LDs from the ER, potentially functioning together with tether or motor proteins to enable subsequent LD trafficking to other cellular locations. To test this hypothesis, we will mechanistic dissection of the Vps45’s role in licensing LD motility within the yeast ER network.
Research methodology: Our approach integrates advanced microscopy, biochemistry, and genetics to elucidate the Vps45's role in transitioning LDs from a static to a motile state. We will assess potential functional relationships between Vps45 and known LD tether protein Mdm1, and the motor protein Myo2 – by determining their localization relative to Erg6-marked LDs in wt and vps45Δ yeast cells using advanced fluorescence microscopy. Mistargeting of any of these proteins in vps45Δ cells will prompt co-immunoprecipitation (Co-IP) studies, followed by confirmation of interactions through mutant analysis. In parallel, We will identify novel Vps45 interactors using affinity purification-mass spectrometry with GFP- or TurboID-tagged Vps45, validate candidates biochemically, and determine their functional roles in LD mobility.
Supervisor
GAO, Jieqiong
Quota
1
Course type
UROP1100
Applicant's Roles
Our lab is seeking a motivated undergraduate student to join our research team as an Introductory Research Assistant. This position is ideal for students with limited research experience who are eager to learn fundamental scientific techniques and contribute to ongoing research projects. The successful applicant will gain valuable hands-on laboratory experience.
Applicant's Learning Objectives
This research experience will provide undergraduate students with opportunities to develop critical scientific thinking and essential laboratory skills. Participants will:
(1) Master Core Laboratory Techniques: Students will gain hands-on experience applying the scientific method, including yeast strain construction, verification of protein expression via Western blot analysis, protein purification from yeast, and in vitro pulldown assays.
(2) Cultivate Research & Analytical Skills: Students will develop proficiency in literature searching, critical review of scientific publications, and principles of data management and organization.
(3) Understand the Scientific Process: Students will apply the scientific method directly to our ongoing research project, contributing to experimental design, data analysis, and interpretation.
(1) Master Core Laboratory Techniques: Students will gain hands-on experience applying the scientific method, including yeast strain construction, verification of protein expression via Western blot analysis, protein purification from yeast, and in vitro pulldown assays.
(2) Cultivate Research & Analytical Skills: Students will develop proficiency in literature searching, critical review of scientific publications, and principles of data management and organization.
(3) Understand the Scientific Process: Students will apply the scientific method directly to our ongoing research project, contributing to experimental design, data analysis, and interpretation.
Complexity of the project
Easy