Microtubules Stabilization by Mutant Spastin Affects ER Morphology and Ca2+ Handling

Abstract

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca 2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed.

In vivo Analysis of CRISPR/Cas9 Induced Atlastin Pathological Mutations in Drosophila

“…Thus, the severity of the CRISPR homozygous mutants suggests a graded loss of function effect, confirming the results obtained from the previous experiments. We note that average ER profile length in controls (w 1118 stock), while consistent with previous reports ( Orso et al, 2009 ; Espadas et al, 2019 ; Vajente et al, 2019 ), is shorter than in some mutants as if mutants had a greater baseline profile length ( Figures 4B,C ). However, because the graded effect of the mutations is solid and all lines are grown under the same environmental conditions, we hypothesize that this could be due to the potentially very different genetic background between w 1118 controls and mutants ( Chandler et al, 2013 ).…”

Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress

“…Atlastin1, a transmembrane protein with GTPase activity associated with SPG3A, drives the generation of ER three-way junctions ( Hu et al, 2009 ; Orso et al, 2009 ; Pendin et al, 2011 ) and modifies the ER morphology by homotypic membrane fusion. In addition to these HSP proteins participating in membrane shaping, spastin, which is mutated in SPG4, has been found to disassemble and remodel neuronal microtubules and to maintain ER structure integrity and calcium homeostasis ( Orso et al, 2005 ; Evans et al, 2006 ; Sanderson et al, 2006 ; Vajente et al, 2019 ).…”

Lighting Up Ca2+ Dynamics in Animal Models

“…There are two major families of indicators that have been developed, i.e., chemical probes and GECIs. Chemical indicators (e.g., fura-2, indo-1, fluo-4), small fluorescent molecules based on BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid) chelating moiety, are widely used in cultured and isolated cells [25][26][27] but have been poorly exploited in live animal models [28][29][30][31], which is mainly due to tissue permeation issues. On the contrary, GECIs have been widely exploited in live animals due to the techniques available to express the engineered Ca 2+ sensing proteins that they encode in vivo [32][33][34].…”

“…The ease of genetic manipulation in flies allows the combination of Ca 2+ imaging with other genetically encoded tools, e.g., optogenetics [129][130][131] and genetically encoded voltage indicators (GEVIs) [132,133]. GECIs expressed in flies can also be exploited to perform in vitro, i.e., primary neuronal culture [26,27,134], or ex vivo Ca 2+ imaging experiments, e.g., larval neuromuscular preparation [36] or the isolated larval brain [135].…”