Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology.
Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations.
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA.
Guohua
G
Zhao
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA.
Peng-Peng
PP
Zhu
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA.
Benoît
B
Renvoisé
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA.
Lymarie
L
Maldonado-Báez
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA.
Seong Hee
SH
Park
Cell Biology Section, Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892-3738, USA. Electronic address: blackstc@ninds.nih.gov.
Craig
C
Blackstone
D017667:Adipocytes / Q000166:cytology; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002453:Cell Cycle; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D008322:Mammals; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D010641:Phenotype; D015398:Signal Transduction; D014759:Viral Envelope Proteins / Q000378:metabolism
D019485:Bone Morphogenetic Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D020558:GTP Phosphohydrolases
CRISPR/Cas9; Endoplasmic reticulum; GTPase; Hereditary spastic paraplegia; Lipid droplet; Morphology