Dynein-mediated microtubule translocation powering neurite outgrowth in chick and Aplysia neurons requires microtubule assembly

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line.

The Journal of Cell Biology ◽

10.1083/jcb.100.3.764 ◽

1985 ◽

Vol 100 (3) ◽

pp. 764-774 ◽

Cited By ~ 144

Author(s):

D L Gard ◽

M W Kirschner

Keyword(s):

Neurite Outgrowth ◽

Isoelectric Focusing ◽

Developmental Regulation ◽

Neuroblastoma Cell ◽

Microtubule Assembly ◽

Beta Tubulin ◽

Fourfold Increase ◽

Cell Extracts ◽

Mouse Neuroblastoma ◽

Sds Page

We have examined the phosphorylation of cellular microtubule proteins during differentiation and neurite outgrowth in N115 mouse neuroblastoma cells. N115 differentiation, induced by serum withdrawal, is accompanied by a fourfold increase in phosphorylation of a 54,000-mol-wt protein identified as a specific isoform of beta-tubulin by SDS PAGE, two-dimensional isoelectric focusing/SDS PAGE, and immunoprecipitation with a specific monoclonal antiserum. Isoelectric focusing/SDS PAGE of [35S]methionine-labeled cell extracts revealed that the phosphorylated isoform of beta-tubulin, termed beta 2, is one of three isoforms detected in differentiated N115 cells, and is diminished in amounts in the undifferentiated cells. Taxol, a drug which promotes microtubule assembly, stimulates phosphorylation of beta-tubulin in both differentiated and undifferentiated N115 cells. In contrast, treatment of differentiated cells with either colcemid or nocodazole causes a rapid decrease in beta-tubulin phosphorylation. Thus, the phosphorylation of beta-tubulin in N115 cells is coupled to the levels of cellular microtubules. The observed increase in beta-tubulin phosphorylation during differentiation then reflects developmental regulation of microtubule assembly during neurite outgrowth, rather than developmental regulation of a tubulin kinase activity.

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Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors.

The Journal of Cell Biology ◽

10.1083/jcb.101.5.1799 ◽

1985 ◽

Vol 101 (5) ◽

pp. 1799-1807 ◽

Cited By ~ 338

Author(s):

D G Drubin ◽

S C Feinstein ◽

E M Shooter ◽

M W Kirschner

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Time Course ◽

Microtubule Assembly ◽

Nerve Growth ◽

Peripheral Neurons ◽

Associated Proteins

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.

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GIT1 enhances neurite outgrowth by stimulating microtubule assembly

Neural Regeneration Research ◽

10.4103/1673-5374.179054 ◽

2016 ◽

Vol 11 (3) ◽

pp. 427 ◽

Cited By ~ 9

Author(s):

Hai-lian Shen ◽

Wei-Qiang Gao ◽

Yi-sheng Li ◽

Li-xia Qin ◽

Jie Liu ◽

...

Keyword(s):

Neurite Outgrowth ◽

Microtubule Assembly

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Sense and antisense transfection analysis of tau function: tau influences net microtubule assembly, neurite outgrowth and neuritic stability

Journal of Cell Science ◽

10.1242/jcs.107.4.869 ◽

1994 ◽

Vol 107 (4) ◽

pp. 869-879 ◽

Cited By ~ 12

Author(s):

B. Esmaeli-Azad ◽

J.H. McCarty ◽

S.C. Feinstein

Keyword(s):

Neurite Outgrowth ◽

Neuronal Cell ◽

Microtubule Assembly ◽

Cell Behavior ◽

Dna Transfection ◽

Stable Transfectants ◽

Protein Map ◽

Direct Support

Microtubules are fundamental elements participating in many aspects of cell behavior and maintenance, yet the factors regulating microtubule behavior in vivo remain poorly understood. Employing the nerve growth factor (NGF)-responsive cell line, PC12, we have used sense and antisense DNA transfection strategies to examine the role of the microtubule-associated protein (MAP) tau in several aspects of neuronal cell behavior. Stable transfectants over-expressing tau accumulate more microtubule mass and extend neurites more rapidly than control cells, while transfectants under-expressing tau exhibit reduced microtubule levels and slower neurite outgrowth. Further, tau over-expressing cells are markedly more resistant to nocodazole-induced neuritic degeneration when compared to wild-type or tau under-expressing cells. These observations provide direct support for the model that tau is capable of influencing: (i) net microtubule assembly, (ii) the rate of neurite elongation and (iii) neuritic stability. These capabilities suggest that tau plays crucial roles in the development and maintenance of neuronal cells.

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Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166117 ◽

1996 ◽

Vol 54 ◽

pp. 730-731

Author(s):

E. D. Salmon ◽

J. C. Waters ◽

C. Waterman-Storer

Keyword(s):

Imaging System ◽

Ccd Camera ◽

Transmitted Light ◽

Microtubule Assembly ◽

Live Cells ◽

Optical Efficiency ◽

Multiple Band ◽

Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

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