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. 2021 Mar 1;32(5):422-434.
doi: 10.1091/mbc.E19-09-0530. Epub 2021 Jan 6.

Doublecortin facilitates the elongation of the somatic Golgi apparatus into proximal dendrites

Affiliations

Doublecortin facilitates the elongation of the somatic Golgi apparatus into proximal dendrites

Peijun Li et al. Mol Biol Cell. .

Abstract

Mutations in the doublecortin (DCX) gene, which encodes a microtubule (MT)-binding protein, cause human cortical malformations, including lissencephaly and subcortical band heterotopia. A deficiency in DCX and DCX-like kinase 1 (DCLK1), a functionally redundant and structurally similar cognate of DCX, decreases neurite length and increases the number of primary neurites directly arising from the soma. The underlying mechanism is not completely understood. In this study, the elongation of the somatic Golgi apparatus into proximal dendrites, which have been implicated in dendrite patterning, was significantly decreased in the absence of DCX/DCLK1. Phosphorylation of DCX at S47 or S327 was involved in this process. DCX deficiency shifted the distribution of CLASP2 proteins to the soma from the dendrites. In addition to CLASP2, dynein and its cofactor JIP3 were abnormally distributed in DCX-deficient neurons. The association between JIP3 and dynein was significantly increased in the absence of DCX. Down-regulation of CLASP2 or JIP3 expression with specific shRNAs rescued the Golgi phenotype observed in DCX-deficient neurons. We conclude that DCX regulates the elongation of the Golgi apparatus into proximal dendrites through MT-associated proteins and motors.

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Figures

FIGURE 1:
FIGURE 1:
The dendritic localization of the Golgi is impaired in DCX-deficient neurons. (A) Cultured hippocampal neurons from WT or Dcx-/y and Dclk1–/– single and double mutant mice were stained for GM130 (purple), tubulin (green), and DAPI (blue) on DIV4. A WT neuron has a somatic Golgi apparatus (GM130 staining) extended into the dendrite. In the Dcx/Dclk1 mutant neurons, most of the Golgi apparatus did not extend into the dendrite. The scale bar represents 10 µm. (B) Schematic of the measurement of the distance from the somatic Golgi apparatus or centrosome to the nucleus: the distances were measured from the distal edge of the somatic Golgi apparatus or the center of the centrosome to the closest edge of the nucleus, and indicated as “a” or “b,” respectively. (C) The distances of the somatic Golgi apparatus to the nucleus were quantified and compared among WT (n = 189), Dcx-/y (n = 213), Dclk1–/– (n = 156), or Dcx-/y;Dclk1–/– (n = 163) neurons. (D) The centrosome remained associated with the Golgi in Dcx/Dclk1 mutant neurons. The scale bar represents 10 µm. (E) Similar to the Golgi, the translocation of the centrosome into the primary dendrite was impaired in Dcx/Dclk1 mutant neurons. (F) Representative electron microscopy images of the WT and Dcx-/y;Dclk1–/– cortex showing Golgi structure (indicated with arrows). Data were obtained from four independent experiments. *P < 0.05; #P < 0.01; P values were calculated from t tests.
FIGURE 2:
FIGURE 2:
Rescue mediated by shRNA reveals the importance of DCX phosphorylation on Golgi outpost formation. (A) Cultured hippocampal cells from embryonic day 18 (E18) mice were transfected with vectors expressing GFP and a scrambled control shRNA (A); GFP and DCX shRNA (B); GFP, DCX shRNA, and full-length DCX tagged with HA (DCX shRNA rescue) on DIV 3 (C). Three days after transfection (DIV6), cells were immunostained for the Golgi (GM130) and DCX. Arrowheads indicate the location of the Golgi apparatus and the edge of the nucleus. The scale bar represents 10 μm. (D) The schematic of the rescue construct is shown with residues of interest for subsequent studies. (E) The distance of the Golgi to the nuclear envelope was measured and quantified in transfected neurons (n = 220, 198, 176 for control, Dcx shRNA, and Dcx rescue, respectively). The expression of full-length DCX in DCX-deficient neurons rescues the phenotype of impaired Golgi extension into dendrites. The scale bar represents 10 μm. (F) Plasmids expressing nonphosphorylatable DCX mutants S47A, S297A or S327A were cotransfected with plasmids expressing GFP and the DCX shRNA on DIV3, and cells were stained for GM130 on DIV6. Distances of somatic Golgi to the nucleus in each rescue experiment were measured and compared with cells transfected with the DCX shRNA. Both DCX and DCX S297A rescued the Golgi phenotype, but not DCX S327A and S47A. Furthermore, DCX S327A further decreased the distance of the somatic Golgi to the nucleus, suggesting a dominant negative effect. Data were obtained from three independent experiments, and more than 150 neurons (n = 220, 198, 176, 192, 159, and 210 for each group) were analyzed. *P < 0.05, #P < 0.01, P values were calculated from t tests.
FIGURE 3:
FIGURE 3:
Loss of DCX decreases CLASP2 localization in dendrites. (A–C) Cultured WT hippocampal neurons were fixed on DIV5 and immunostained for DCX, CLASP2, and GM130, as indicated. (A) DCX and CLASP2 were distributed similarly in the neuronal soma and distal dendrites. (B) Dual immunostaining for DCX and GM130. (C) Dual immunostaining for DCLASP2 and GM130. (D) Cultured hippocampal neurons were transfected with plasmids expressing GFP and a control (scrambled) shRNA or DCX shRNA on DIV3. Transfected neurons were fixed on DIV6 and immunostained for DCX and CLASP2. GFP-positive cells were transfected with shRNA constructs that also express GFP. The CLASP2 intensity was quantified along the trajectories of neural processes starting from the soma and extending out 50 μm (shown as a broken white line adjacent to the neurite). White or purple arrows indicate DCX staining in transfected neurons or nontransfected neurons, respectively. (E) The intensity of CLASP2 immunostaining was measured in the soma of control or Dcx KD neurons. Significantly higher CLASP2 levels were detected in the soma of mutants (n = 42) than in controls (n = 47). (F) CLASP2 intensities in neural processes from control (n = 47) or DCX KD (n = 42) neurons were quantified from the soma and extending to 50 μm. (G) CLASP2 knockdown with CLASP2 shRNA-A rescued the Golgi phenotype observed in DCX-deficient neurons. The results were obtained from three independent experiments and 140 neurons from each condition. #P < 0.01, P values were calculated from the t test. The scale bar represents 10 μm.
FIGURE 4:
FIGURE 4:
Molecular motors and the somatic Golgi dendritic distribution (A) Cultured WT hippocampal neurons (DIV5) were stained with the Golgi marker giantin and kinesin-3. (B) Down-regulation of kinesin-3 with the kinesin-3 shRNA decreased the dendritic distribution of the Golgi compared with the control. Arrows indicate the locations of Golgi stained with GM130. (C) The distances of the somatic Golgi to the nucleus were quantified and compared. Data were obtained from three independent experiments, and 90 neurons were analyzed from each condition. (D) Cultured hippocampal neurons (DIV5) from WT or Dcx-/y; Dclk1–/– mice were stained with GM130 and DHC antibodies. (E) The colocalization between dynein and GM130 in cultured neurons from WT or Dcx-/y; Dclk1–/– was calculated using the “Colocalization Threshold” plugin of the ImageJ software platform. Only GM130-stained regions were outlined and analyzed for colocalization. Pearson’s correlation coefficient (Rcolocalization) and Mander’s colocalization coefficient (M1 and M2) calculated from the analysis are shown in the table. Rcolocalization is defined as Pearson’s correlation coefficient for pixels where both the red channel (dynein) and green channel (GM130) are above their respective thresholds. M1 and M2 are Mander’s colocalization coefficients obtained using thresholds. Data are based on three independent experiments from each condition. P values were calculated from t tests. *P < 0.05, #P < 0.01. Scale bars represent 10 μm.
FIGURE 5:
FIGURE 5:
The dynein cofactor JIP3 is involved in the effects of DCX on the dendritic distribution of the Golgi. (A) Immunostaining for JIP3 and DHC in cultured neurons from WT and Dcx-/y P0 mice on DIV5. (B) Immuno­precipitation of DIC and Western blot showing JIP3 levels in protein lysates from P0 WT or Dcx-/y mouse brains. (C) Quantification of Western blot bands for JIP3 pulled down with a DIC antibody normalized to JIP3 inputs. The results were obtained from three independent experiments. (D) Three JIP3 shRNAs were validated for their ability to down-regulate JIP3 expression. Different groups of HEK293 cells were transfected with a plasmid overexpressing JIP3 (lane 1) or plasmid overexpressing JIP3 and plasmid expressing one of the three JIP3 shRNAs (lanes 2–4). (E) Represen­tative images showing the effects of JIP3 on the somatic Golgi (GM130) distribution. Cultured neurons were transfected with different plasmids to modify gene expression: control (control shRNA), JIP3 KD (JIP3 shRNA#1), DCX KD (DCX shRNA), JIP3 (overexpressing JIP3), C-JIP3 (overexpressing C-JIP3), or their combinations on DIV4. The dendritic distribution of the Golgi apparatus was observed by performing GM130 immunostaining on DIV 6. Scale bars represent 10 μm. (F) The distances from the Golgi to the nuclear envelope were measured and analyzed for cells from each condition. Data were obtained from three independent experiments. The number of neurons analyzed in each group is shown in each bar. *P < 0.05, compared with control group; #P < 0.05, compared with DCX KD group, P values were calculated from t tests.
FIGURE 6:
FIGURE 6:
Schematic diagram shows that DCX facilitates the extension of the somatic Golgi complex into dendrite through MT-associated proteins and motors. In WT neurons, DCX association with kinesin-3 helps kinesin–3-mediated anterograde transports (Liu et al., 2012). Meanwhile, DCX binds dynein while inhibiting JIP3. We hypothesize that DCX negatively regulates dynein-mediated retrograde transports. The combination of DCX effects on retrograde and anterograde transports guarantee the normal extension of somatic Golgi complex into dendrites. In DCX KO neurons (without DCX), more CLASP2 and kinesin-3 proteins are restricted in neuronal soma. Kinesin–3-mediated anterograde transports are decreased without DCX (Liu et al., 2012). More JIP3 molecules bind dynein. JIP3 needs to bind an adaptor protein before associates with dynein. We hypothesize that dynein-mediated retrograde transports are increased with JIP3 association while not bound with DCX. The balance between anterograde transports and retrograde transports is changed without DCX, which results in less Golgi extension into dendrites.

References

    1. Akhmanova A, Hoogenraad CC, Drabek K, Stepanova T, Dortland B, Verkerk T, Vermeulen W, Burgering BM, De Zeeuw CI, Grosveld F, et al. (2001). Clasps are CLIP-115 and -170 associating proteins involved in the regional regulation of microtubule dynamics in motile fibroblasts. Cell 104, 923–935. - PubMed
    1. Akhmanova A, Severin F (2004). Thirteen is the lucky number for doublecortin. Dev Cell 7, 5–6. - PubMed
    1. Amano M, Tsumura Y, Taki K, Harada H, Mori K, Nishioka T, Kato K, Suzuki T, Nishioka Y, Iwamatsu A, et al. (2010). A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase. PLoS One 5, e8704. - PMC - PubMed
    1. Arimoto M, Koushika SP, Choudhary BC, Li C, Matsumoto K, Hisamoto N (2011). The Caenorhabditis elegans JIP3 protein UNC-16 functions as an adaptor to link kinesin-1 with cytoplasmic dynein. J Neurosci 31, 2216–2224. - PMC - PubMed
    1. Arthur AL, Yang SZ, Abellaneda AM, Wildonger J (2015). Dendrite arborization requires the dynein cofactor NudE. J Cell Sci 128, 2191–2201. - PMC - PubMed

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