Dataset Information

A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

ABSTRACT: We have previously shown that in the developing trunk of zebrafish embryos, two-pore channel type 2 (TPC2)-mediated Ca²⁺ release from endolysosomes plays a role in the formation of the skeletal slow muscle. In addition, TPC2-mediated Ca²⁺ signaling is required for axon extension and the establishment of synchronized activity in the primary motor neurons. Here, we report that TPC2 might also play a role in the development of the notochord of zebrafish embryos. For example, when tpcn2 was knocked down or out, increased numbers of small vacuoles were formed in the inner notochord cells, compared with the single large vacuole in the notochord of control embryos. This abnormal vacuolation was associated with embryos displaying attenuated body axis straightening. We also showed that TPC2 has a distinct pattern of localization in the notochord in embryos at ∼24 hpf. Finally, we conducted RNAseq to identify differentially expressed genes in tpcn2 mutants compared to wild-type controls, and found that those involved in actin filament severing, cellular component morphogenesis, Ca²⁺ binding, and structural constituent of cytoskeleton were downregulated in the mutants. Together, our data suggest that TPC2 activity plays a key role in notochord biogenesis in zebrafish embryos.

SUBMITTER: Rice KL

PROVIDER: S-EPMC10658360 | biostudies-literature | 2023 Jan-Dec

REPOSITORIES: biostudies-literature

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A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

Rice Keira L KL Chan Ching Man CM Kelu Jeffrey J JJ Miller Andrew L AL Webb Sarah E SE

Contact (Thousand Oaks (Ventura County, Calif.)) 20230101

We have previously shown that in the developing trunk of zebrafish embryos, two-pore channel type 2 (TPC2)-mediated Ca<sup>2+</sup> release from endolysosomes plays a role in the formation of the skeletal slow muscle. In addition, TPC2-mediated Ca<sup>2+</sup> signaling is required for axon extension and the establishment of synchronized activity in the primary motor neurons. Here, we report that TPC2 might also play a role in the development of the notochord of zebrafish embryos. For example, w ...[more]

PMID: 38028019

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Project description:Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Dataset Information

A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

Publications

A Role for Two-Pore Channel Type 2 (TPC2)-Mediated Regulation of Membrane Contact Sites During Zebrafish Notochord Biogenesis?

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