Loss of all 3 Extended Synaptotagmins does not affect normal mouse development, viability or fertility

Abstract

The extended synaptotagmins, E-Syt1, 2 and 3, are multiple C2 domain membrane proteins that are tethered to the endoplasmic reticulum and interact in a calcium dependent manner with plasma membrane phospholipids to form endoplasmic reticulum - plasma membrane junctions. These junctions have been implicated in the exchange of phospholipids between the 2 organelles. The E-Syts have further been implicated in receptor signaling and endocytosis and can interact directly with fibroblast growth factor and other cell surface receptors. Despite these multiple functions, the search for a requirement in vivo has been elusive. Most recently, we found that the genes for E-Syt2 and 3 could be inactivated without effect on mouse development, viability, fertility or morphology. We have now created insertion and deletion mutations in the last of the mouse E-Syt genes. We show that E-Syt1 is specifically expressed throughout the embryonic skeleton during the early stages of chrondrogenesis in a pattern quite distinct from that of E-Syt2 or 3. Despite this, E-Syt1 is also not required for mouse development and propagation. We further show that even the combined inactivation of all 3 E-Syt genes has no effect on mouse viability or fertility in the laboratory. However, this inactivation induces an enhancement in the expression of the genes encoding Orp5/8, Orai1, STIM1 and TMEM110, endoplasmic reticulum - plasma membrane junction proteins that potentially could compensate for E-Syt loss. Given the multiple functions suggested for the E-Syts and their evolutionary conservation, our unexpected findings suggest that they may only provide a survival advantage under specific conditions that have as yet to be identified.

Feedback regulation of insulin secretion by extended synaptotagmin‐1

“…E‐Syt1 and E‐Syt2 are ubiquitously expressed in humans, whereas E‐Syt3 is mainly found in the nervous system (The Human Protein Atlas) (29, 30). Immunoblotting of mouse tissue confirmed the selective expression of E‐Syt3 in the brain, whereas E‐Syt1 and E‐Syt2 were detected in most tissues tested.…”

“…In this study, we find that E‐Syt1 acts as a negative feedback regulator of glucose‐stimulated insulin secretion by clearing the PM of DAG formed after insulin granule exocytosis. In previous work, it was shown that mice lacking all 3 E‐Syt isoforms are viable and exhibit no overt defects, although compensatory up‐regulation of other components of ER‐PM contacts was observed and may complicate the interpretation (29, 30). HeLa cells lacking all 3 E‐Syt isoforms exhibit reduced ER‐PM contact densities but show no major defects in overall lipid handling or Ca 2+ homeostasis (6).…”

The Extended-Synaptotagmins

“…In mammals, E-Syts have broad tissue distribution [36]. Surprisingly, their absence does not have a major disrupting effect on cellular life, as tricalbin triple knockout yeast cells and E-Syt triple knockout mammalian cells are viable [5, 6, 30].…”

“…Surprisingly, their absence does not have a major disrupting effect on cellular life, as tricalbin triple knockout yeast cells and E-Syt triple knockout mammalian cells are viable [5, 6, 30]. Additionally, mutant mice lacking all the three E-Syts develop normally, are viable and fertile [36, 37]. Thus, while it was reported that E-Syt2 is required for rapid endocytosis and signaling of growth factor receptors, including Fibroblast Growth Factor (FGF) receptor, such role is clearly dispensable in live animals [38–40].…”

ER-plasma membrane junctions: Why and how do we study them?

“…Reconstitutions of E-Syt1 and E-Syt2 in triple knockdown cells were sufficient to restore the abundance of ER-PM junctions to the control level, indicting the redundancy of E-Syts in ER -PM tethering. Triple E-Syt knockout animals are viable without obvious defects [136, 137]. These findings indicate that there are tethering mechanisms besides E-Syts maintaining ER-PM junctions.…”