Based on the observations that (i) γ-BAR directly interacts with conventional kinesins (see above), (ii) upon overexpression associates with highly mobile membrane carriers throughout the cell (timelapse movies and (Neubrand et al., 2005)) and (iii) the corresponding shift of perinuclear REs towards the cell periphery, we speculate that γ- BAR is involved in the transport and/ or intracellular positioning of these organelles.
Results 75 Thus, the accumulation of REs at the cell periphery upon γ-BAR overexpression should depend on microtubules and associated motor proteins like conventional kinesins. To test this, we treated HeLa cells with the microtubule de-polymerizing agent nocodazole and analyzed the distribution of transfected γ-BAR-eGFP and endogenous kinesin.
Figure 3-22: Nocodazole treatment disperses the peripherally accumulated compartment
HeLa cells were transfected with constructs encoding γ-BAR-eGFP, treated with 10 µM nocodazole for 1 hour at 37°C (lower panel) or left untreated (control, upper panel), fixed and stained for endogenous conventional kinesin using antibodies against ubiquitous kinesin heavy chains (HC). DAPI-stained nuclei are shown in blue. Insets represent 3-fold magnification of boxed areas. The scale bar is 10 µm.
Conventional kinesin accumulated together with γ-BAR-eGFP in the peripheral compartment (Figure 3-22, upper panel). This accumulation is quite striking since non- transfected HeLa cells did not display a similar staining pattern when using the kinesin antibody at the same dilution (data not shown). Following treatment with nocodazole, the peripheral compartment got dispersed into smaller discrete membrane structures containing γ-BAR-eGFP. Kinesin remained associated with these structures. Additionally, we examined the distribution of AP-1 and TfR in γ-BAR-eGFP expressing cells after nocodazole treatment (Figure 3-23). Also these two marker proteins were associated with the dispersed compartment.
These results demonstrate that the accumulation and positioning of the peripheral compartment is dependent on microtubules and perhaps on the activity of microtubule- associated motor proteins, i.e. conventional kinesins.
Figure 3-23: TfR and AP-1 stay associated with the dispersed compartment after nocodazole treatment
γ-BAR-eGFP expressing HeLa cells were treated as described in Figure 3-22 and stained for endogenous transferrin receptor (upper panel) and AP-1 (lower panel). Insets show 3-fold magnified view of boxed areas. DAPI-stained nuclei are shown in blue. The scale bar is 10 µm.
In order to analyze whether the γ-BAR driven accumulation of the RE compartment is indeed dependent on conventional kinesin, we treated HeLa cells with two different pre- designed siRNAs (Qiagen) against the ubiquitous kinesin-1 heavy chain family member KIF5B and analyzed the distribution of co-transfected γ-BAR-eGFP in these cells. Knockdown efficiency was controlled by immunostaining for endogenous conventional kinesin heavy chain. Knockdown of KIF5B resulted in a significantly higher degree of perinuclear accumulation of γ-BAR-eGFP-containing organelles. 86% or 90% of the siRNA treated cells displayed a perinuclear accumulation of γ-BAR, whereas only 12% of the control siRNA treated cells contained perinuclear γ-BAR-eGFP (Figure 3-24).
Results 77
Figure 3-24: Intact kinesin KIF5 is necessary for the γ-BAR induced re-localization of REs
(A) HeLa cells were treated twice with siRNAs against the ubiquitous kinesin heavy chain KIF5B or a
control siRNA (day 1 and 4) and co-transfected with expression plasmids encoding γ-BAR-eGFP (day 4). On day 5 cells were fixed and immunostained using antibodies against conventional kinesin heavy chain (HC). KIF5B siRNA-treated cells were devoid of kinesin immunoreactivity and displayed a high degree of perinuclear accumulated γ-BAR-eGFP. Insets represent 3-fold magnification of boxed areas. DAPI- stained nuclei are shown in blue. The scale bar is 10 µm.
(B) Quantification of the effects exemplified in (A). The fraction of γ-BAR-eGFP transfected cells
containing perinuclear accumulated γ-BAR was plotted. Two different siRNAs against KIF5B gave almost identical results. 50 cells were scored for each data point.
Using co-immunoprecipitation experiments we could show that an amino-terminal fragment of γ-BAR comprising amino acids 1-140 does not bind to kinesin (see Figure 3-16), but maintains its membrane association due to palmitoylation. We sought to investigate the subcellular distribution of this construct and its effect on the RE marker protein TfR and on conventional kinesin. We therefore transfected HeLa cells with an eGFP-tagged version of γ-BAR (aa 1-140) and analyzed fixed cells with antibodies against endogenous TfR and kinesin HC (Figure 3-25).
The amino-terminal fragment of γ-BAR localized to membranes in a perinuclear compartment but also to discrete endosomally-like structures dispersed throughout the cytoplasm. These structures strikingly resemble the dispersed organelles visible after nocodazole treatment (see Figure 3-22 and Figure 3-23). γ-BAR (aa 1-140)-eGFP containing structures were immuno-positive for TfR but did not contain kinesin. Thus, it appears that the carboxy-terminal domain of γ-BAR facilitates recruitment of conventional kinesin to membranes and that this interaction is crucial for the peripheral accumulation of REs. Disruption of microtubules or transfection of a kinesin binding- deficient γ-BAR mutant led to a dispersed distribution of TfR positive REs.
Figure 3-25: γ-BAR (aa 1-140) co-localizes with TfR but not with kinesin
HeLa cells were transfected with plasmids encoding eGFP-tagged γ-BAR (aa 1-140), fixed and processed for immuno-fluorescence staining using antibodies against transferrin receptor (upper panel) and kinesin HC (lower panel). DAPI-stained nuclei are shown in blue. Insets represent 3-fold magnification of boxed areas. The scale bar is 10 µm.
Results 79
3.5.4 Conventional kinesin KIF5 is necessary for the positioning of