GMAP-210 Is Not Associated With γ Tubulin

In Rios et al. (2004) GMAP-210 was shown to recruit γ-tubulin containing complexes (γ-TCCs) to the Golgi apparatus of Cos-7 cells. Overexpression of a full-length construct of GMAP-210 caused strong staining for γ-tubulin on the Golgi. A construct missing the C-terminus did not cause such an effect. Due to the confusion about the interaction and targeting domains of GMAP-210 in the literature (compare Infanteet al.(1999) and Gillinghamet al.(2004) and 2.1.2, p.29ff.) it was verified if this effect could be reproduced with the described or a different domains of GMAP-210.

HeLa L cells were transfected for 24 h with GFP-tagged constructs for GMAP-210 N-terminus (pEGFPC2-GMAP-210 1-375 and pEGFPC2-GMAP-210 1-1712), GMAP- 210 full-length or GMAP-210 C-terminus (pEGFPC2-GMAP-210 1598-1979). The cells were fixed and stained forγ-tubulin and analysed by epifluorescence microscopy (Fig.45, p.78, right panel). As positive control the N-terminal 702 amino acids of the ninein-like

Figure 52: Golgi Positioning Near The Centrosome After GMAP-210 Depletion. HeLa L cells were transfected with siRNA oligos against GL2 (control) or GMAP-210 3’-UTR for 72 h and fixed with methanol; cells were stained with mouse anti CNAP-1 (red, 1:1000) and sheep anti GM130 (green, 1:500) antibodies; blue channel: DAPI staining; Bar=5μm.

protein (NLP) were transfected into HeLa L cells (gift from M. Casenghi). NLP was described to recruit γ-tubulin ring complexes (γ-TuRCs) upon overexpression (Casenghi

et al., 2003).

In untransfected cells, γ-tubulin staining could be found on two dots in the perinuclear region, the centrosomes of G2 phase cells. Also diffuse cytoplasmic staining could be seen. Cells transfected with the N-terminus of NLP could easily be identified by aggregates of GFP, which at the same time stained for γ-tubulin. When GMAP-210 full-length was transfected, GFP stained the Golgi apparatus. γ-tubulin still localised to the centrosomes and could not be found co-localising with GMAP-210. The Golgi apparatus still localised near the centrosome as normal. Overexpression of either N-terminus or C-terminus did also not recruit γ-tubulin to the Golgi apparatus in HeLa L cells. As a side note, over- expressed NLP did not disturb the microtubule network of interphase cells (Fig.45, p.78 bottom left).

It was furthermore tested if the loss of GMAP-210 in HeLa L cells disturbed theγ-tubulin distribution or positioning of the Golgi apparatus in relation to the microtubule organis- ing centre. For this, HeLa L cells were treated with siRNA against GMAP-210 or GL2 control for 72 h, fixed and either co-stained for GMAP-210 andγ-tubulin (Fig.46, p.79) or GM130 and the centrosomal protein CNAP1 (Fig.52). After depletion of GMAP-210 the antibody against γ-tubulin still stained two dots in the perinuclear region as expected. CNAP1 staining on the centrosomes was also unchanged. GM130 staining monitors the Golgi positioning. After GMAP-210 siRNA, the Golgi apparatus compacted (see 2.3, p.48) but remained adjacent to the CNAP1 staining in the perinuclear region. Golgi po- sitioning near the centrosome was not dependent on GMAP-210.

If GMAP-210 interacted with γ-tubulin, then a high concentration of γ-tubulin in the cell, should recruit at least some GMAP-210. γ-tubulin recruitment could be achieved by overexpression of the NLP N-terminus (Casenghiet al., 2003), as shown in Fig.45 (p.78).

Figure 53: GMAP-210 Localisation In NLP Transfected Cells. HeLa L cells were transfected with a construct for expression of N-terminally GFP-tagged NLP (green) for 24 hours and fixed with paraformaldehyde; cells were stained with rabbit anti GMAP-210 (red, 1:500); blue channel: DAPI staining; Bar=10μm.

GMAP-210 interaction with microtubules was tested by different methods. First the ef- fect of GMAP-210 overexpression and depletion on the microtubule network of HeLa L cells was examined. Overexpression of different N- and C- terminal constructs gave no hint at disturbance of microtubules, neither at their origin, the centrosome, nor in the cell periphery. Slight changes were observed, when a short N-terminal construct (GMAP-210 1-375) was expressed. The microtubules looked more concentrated at the MTOC. Deple- tion of GMAP-210 had also no effect on microtubules.

Recombinant N- and C-terminal protein, purified from Sf9 cells, were tested for their capability to bind to taxol stabilised microtubules. Bundling of microtubules by these proteins was examined. Microtubules incubated with fragments of GMAP-210 were not bundled, in contrast the microtubule binding protein Prc1sp2 easily organised tubules. Microtubules were also incubated with recombinant N- and C-terminus of GMAP-210 and tested in a spin-down assay. In the microtubule pellet no GMAP-210 C-terminus and only little, precipitated N-terminus could be found. Prc1sp2 bound microtubulesin vitro. Furthermore, the effect of GMAP-210 depletion on nocodazole induced Golgi fragmen- tation was tested. In HeLa L cells, Golgi fragments remained more concentrated in the perinuclear region in cells depleted of GMAP-210, if compared to control cells. In

hTERT-RPE1 cells, this effect was not observed. Reassembly of the Golgi after nocodazole washout proceeded with similar kinetics in control cells and cells depleted of GMAP-210.

γ-Tubulin localisation was also unchanged, when GMAP-210, full-length or fragments, were overexpressed. Ninein-like protein, however, recruitedγ-tubulin visibly upon overex- pression, but did not recruit GMAP-210 to these accumulations ofγ-tubulin. GMAP-210 depletion also had no influence on the localisation of γ-tubulin and the Golgi remained near the microtubule organising centre. In any case, γ-tubulin was found on Golgi mem- branes.

2.8

GMAP-210 Recruits The Intraflagellar Transport Protein