Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, indicates its significance as a regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables an efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER reorganized. Knocking out both FAM134B isoforms in myotubes results in aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, indicates its significance as a regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables an efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER reorganized. Knocking out both FAM134B isoforms in myotubes results in aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
Project description:Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise into blastocyst-like structures called blastoids. Naive hPSCs maintenance commonly relies on mouse embryonic fibroblast (MEFs), introducing variability and analytical confounders. Here, we describe a feeder-free culture system based on serum coating that supports long-term naive hPSC maintenance. Across five laboratories, 30 serum batches were evaluated for the expansion of 8 naive hPSC lines for up to 25 passages. Mass spectrometry identified fibronectin and collagens as extracellular matrix proteins consistently present in serum coating. Cells cultured on serum coating displayed growth kinetics, clonogenic capacity, mutation rates, and global gene expression profiles comparable to MEF-based cultures. Importantly, serum-cultured naive hPSCs efficiently underwent germ layer specification, retained trophectoderm competence, and generated blastoids with similar efficiency. Collectively, serum coating provides a scalable, cost-effective, and robust alternative to feeder-based systems, preserving genomic stability and developmental potential while eliminating MEF-associated disadvantages and variability. This platform facilitates large-scale applications of naive hPSCs and enables more reproducible mechanistic studies.
Project description:Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigated the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KO in young mice showed no major phenotypes, however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, led to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibited expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities were observed in cortical ER. Our study unveils critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.
Project description:Using nanoparticles based plasma enrichment strategies on plasma samples, we were able to achieve a huge increase of identified plasma proteins from around 700 to more than 5000 proteins as compared to neat plasma digest. The substantial increase in depth allowed us to perform in-depth analysis and we were able to apply this to a small cohort of plasma samples from pancreatic cancer (PC) patients with primary tumor, tumor that had metastases vs healthy controls. Majority of identified proteins were part of the Human Plasma Proteome Project (HPPP) database, and more than 300 proteins are on the list of FDA approved drug targets. We showed a large and significant increase in ribosomal proteins in plasma of PC that metastasized. ADH1C and ADH1B, both members of the alcohol dehydrogenase family, had one of the largest increase and potentially correlated with liver metastasis. We further highlighted 15 different potentially secreted and/or cell surface proteins that were otherwise not identified in neat, digested plasma and have potential to be useful markers due to their cancer associations. Lastly, we compared different mass spectrometer (Orbitrap Astral and Orbitrap Ascend) and column (depth and throughput) setups on the same dataset and showed similar conclusions potentially can be achieved, although the depth approach on the newer instrumentations can reveal additional insights in the plasma proteome.
Project description:Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, is an important regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER is reorganized. Knocking out both FAM134B isoforms in myotubes results in an aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine-tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.