TY - JOUR
T1 - Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis
AU - Tripathi, Priyanka
AU - Zhu, Zhihui
AU - Qin, Haiyan
AU - Elsherbini, Ahmed
AU - Crivelli, Simone M.
AU - Roush, Emily
AU - Wang, Guanghu
AU - Spassieva, Stefka D.
AU - Bieberich, Erhard
N1 - Publisher Copyright:
© 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.
PY - 2021
Y1 - 2021
N2 - Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.
AB - Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.
KW - Acetylation
KW - Arl13b
KW - Cell biology
KW - Ceramide
KW - Cilia
KW - Lipid rafts
KW - Lipids
KW - Microtubules
KW - Palmitoylation
KW - Tubulin
UR - http://www.scopus.com/inward/record.url?scp=85102625446&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85102625446&partnerID=8YFLogxK
U2 - 10.1194/JLR.RA120001190
DO - 10.1194/JLR.RA120001190
M3 - Article
C2 - 33380429
AN - SCOPUS:85102625446
SN - 0022-2275
VL - 62
JO - Journal of Lipid Research
JF - Journal of Lipid Research
M1 - RA120001190
ER -