TY - JOUR
T1 - Centrosome-dependent microtubule modifications set the conditions for axon formation
AU - Meka, Durga Praveen
AU - Kobler, Oliver
AU - Hong, Shuai
AU - Friedrich, Carina Meta
AU - Wuesthoff, Souhaila
AU - Henis, Melad
AU - Schwanke, Birgit
AU - Krisp, Christoph
AU - Schmuelling, Nessa
AU - Rueter, René
AU - Ruecker, Tabitha
AU - Betleja, Ewelina
AU - Cheng, Tao
AU - Mahjoub, Moe R.
AU - Soba, Peter
AU - Schlüter, Hartmut
AU - Fornasiero, Eugenio F.
AU - Calderon de Anda, Froylan
N1 - Funding Information:
We thank M. Richter (Calderon lab, Zentrum für Molekulare Neurobiologie Hamburg [ZMNH], Universitätsklinikum Hamburg-Eppendorf [UKE]), I. Hermans-Borgmeyer (ZMNH, UKE), and the members of animal facility at the UKE Hamburg for their help with animal experiments. Thanks to the members of Cytometry & Cell Sorting Core Unit, Stem Cell Transplant Clinic, Oncology Center at the UKE for fluorescence-activated cell sorting (FACS) experiments. F.C.A. is supported by Deutsche Forschungsgemeinschaft (DFG) grants FOR 2419, CA1495/1-1, CA 1495/4-1, and CA 1495/7-1; ERA-NET Neuron Grants (Bundesministerium für Bildung und Forschung [BMBF] 01EW1410, 01EW1910, and 01 EW2108B), a JPND Grant (Bundesministerium für Bildung und Forschung 01ED1806), and the University Medical Center Hamburg-Eppendorf (UKE). D.P.M. is a co-applicant in the DFG Grant CA 1495/4-1 for F.C.A. M.H. is partially funded by a scholarship (ID: seventh plan 2012–2017) from the Cultural Affairs and Missions Sector, Ministry of Higher Education of the Arab Republic of Egypt. P.S. is supported by the DFG Heisenberg programm (SO 1337/6-1), DFG (SO 1337/2-2, SO 1337/4-1, SO 1337/7-1), and ERA-NET Neuron (Bundesministerium für Bildung und Forschung 01EW1410 and 01EW1910). E.F.F. is supported by a Schram Stiftung (T0287/35359/2020) and a DFG grant (FO 1342/1-3). F.C.A. conceived the idea and supervised the project. F.C.A. and D.P.M. designed research and interpreted the data. B.S. conducted cell culture work. O.K. and D.P.M. performed STED microscopy. O.K. did the deconvolution. F.C.A. and D.P.M. performed the analysis. D.P.M. and F.C.A. performed in utero electroporation (IUE) surgeries. D.P.M. S.H. C.M.F. N.S. S.W. B.S. and R.R. performed all immunostainings. D.P.M. and N.S. did all epifluorescence imaging. D.P.M. S.H. C.M.F. S.W. N.S. and F.C.A. did the analysis. D.P.M. performed EB3/TACC3 comet live imaging and analysis. D.P.M. did spinning disk imaging and analysis of primary neurons and in situ experiments. E.F.F. performed the long-term time-lapse acquisitions, post hoc SMI 31 immunostainings, and imaging. D.P.M. quantified the data. M.H. performed western blots. E.B. T.C. and M.R.M. developed the Cep120 antibody. B.S. and D.P.M. prepared cells for FACS. H.S. designed and supervised the liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiment; C.K. did the experiments, analysis, and processing; and T.R. assisted with initial analysis. E.F.F. performed the final bioinformatic analysis. F.C.A. and D.P.M. prepared the manuscript with input from all the authors. The authors declare no competing interests.
Funding Information:
We thank M. Richter (Calderon lab, Zentrum für Molekulare Neurobiologie Hamburg [ZMNH], Universitätsklinikum Hamburg-Eppendorf [UKE]), I. Hermans-Borgmeyer (ZMNH, UKE), and the members of animal facility at the UKE Hamburg for their help with animal experiments. Thanks to the members of Cytometry & Cell Sorting Core Unit, Stem Cell Transplant Clinic, Oncology Center at the UKE for fluorescence-activated cell sorting (FACS) experiments. F.C.A. is supported by Deutsche Forschungsgemeinschaft (DFG) grants FOR 2419 , CA1495/1-1 , CA 1495/4-1 , and CA 1495/7-1 ; ERA-NET Neuron Grants ( Bundesministerium für Bildung und Forschung [BMBF] 01EW1410 , 01EW1910 , and 01 EW2108B ), a JPND Grant ( Bundesministerium für Bildung und Forschung 01ED1806 ), and the University Medical Center Hamburg-Eppendorf (UKE) . D.P.M. is a co-applicant in the DFG Grant CA 1495/4-1 for F.C.A. M.H. is partially funded by a scholarship (ID: seventh plan 2012–2017) from the Cultural Affairs and Missions Sector , Ministry of Higher Education of the Arab Republic of Egypt . P.S. is supported by the DFG Heisenberg programm ( SO 1337/6-1 ), DFG ( SO 1337/2-2 , SO 1337/4-1 , SO 1337/7-1 ), and ERA-NET Neuron ( Bundesministerium für Bildung und Forschung 01EW1410 and 01EW1910 ). E.F.F. is supported by a Schram Stiftung ( T0287/35359/2020 ) and a DFG grant ( FO 1342/1-3 ).
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/4/19
Y1 - 2022/4/19
N2 - Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
AB - Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
KW - CP: Cell biology
KW - Cep120
KW - acetylated microtubules
KW - axon formation
KW - centrosome
KW - microtubules
KW - neuronal polarity
UR - http://www.scopus.com/inward/record.url?scp=85128421805&partnerID=8YFLogxK
U2 - 10.1016/j.celrep.2022.110686
DO - 10.1016/j.celrep.2022.110686
M3 - Article
C2 - 35443171
AN - SCOPUS:85128421805
SN - 2211-1247
VL - 39
JO - Cell Reports
JF - Cell Reports
IS - 3
M1 - 110686
ER -