Subcellular localizati on of Arl5b in primary neurons

One way to gain insights into the functi on of a protein is to determine its subcellular localizati on as a starti ng point for further experiments. Based on the expression of Arl5b in the brain during develop-ment and its acti vity regulated expression in the hippocampus (Hermey et al., 2013), fi rst experiments as-sessed subcellular localizati on of Arl5b in dissociated primary cultures of hippocampal neurons. This was achieved by C-terminal fusion of Arl5b with fl uorescent markers and overexpression of the fusion proteins aft er transfecti on. Fluorophores and other tags used to localize the protein were always fused to the C-Terminus of Arl5b, because the N-terminus harbors a consensus site for N-terminal myristoylati on which is shared with other Arf-like proteins and is thought to be important for proper functi on and subcellular localizati on. N-terminal modifi cati ons of Arl5b were investi gated in detail (compare 3.1.3).

Expression of Arl5b-tdTomato in developing hippocampal neurons revealed a ubiquitous distribu-ti on patt ern. In general the subcellular patt ern of Arl5b was assessed with various Arl5b fusion proteins and it did not vary based on the tag fused to Arl5b. For example Arl5b-tdTomato, Arl5b-eGFP, Arl5b-mKate2 and Arl5b-HA exhibit very similar subcellular distributi on in HeLa cells and neurons. Arl5b was found to be present in the neurons soma, dendrites and axon. In this experiment the axon was visualized by coun-terstaining with an axonal marker, an anti body against neurofi lament, and dendrites with the dendriti c marker MAP2 (fi gure 3.2).

Neurofilament Arl5b-tdTomato MAP2

Figure 3.2 Arl5b is localized to the soma, dendrites and axon in principal hippocampal neurons. Cultured hippocampal neurons aft er 4DIV were transfected with Arl5b-tdTomato. One day later neurons were immunostained for Neurofi lament and MAP2, an axonal and a dendriti c marker, respecti vely. Magenta colored regions depict Arl5b located in dendrites and the soma while yel-low color indicates Arl5b distributi on to the axon of developing dissociated hippocampal neurons. The scale bar indicates 20 μm.

Following this fi rst overview the subcellular localizati on of Arl5b was examined in more detail. In the soma a strong accumulati on near the nucleus was prominent. To further characterize the structure hippocampal neurons overexpressing Arl5b-GFP were immuno-stained for two residenti al proteins of the Golgi apparatus, the cis-Golgi marker GM130 and the trans-Golgi maker TGN46. Figure 3.3 shows a strong overlap of the accumulated Arl5b-GFP signal and both Golgi marker proteins.

This experiment revealed that Arl5b is localized to the Golgi apparatus. Due to the dense packing of the stacks in the Golgi apparatus it was diffi cult to assess to which stack of the Golgi the protein is local-ized. One way to address this problem is to disperse the Golgi apparatus. This was achieved by treatment of the cells with Nocodazole. This agent interferes with the polymerizati on of microtubules which is es-senti al for the proper architecture of the Golgi apparatus. However, the polarizati on of the dispersed Golgi vesicles remains preserved. Aft er dispersion each Golgi fragment consists of two polarized parts, a cis- and a trans-side. Arl5b co-localized in these structures with both markers, TGN46 and GM130, aft er dispersion of the Golgi (fi gure 3.4). Therefore Arl5b seems not to be exclusively present in one of these specialized compartments of the Golgi apparatus, but is rather distributed throughout the Golgi network.

Aft er clarifying where Arl5b predominantly resides in the soma, the subpopulati on of Arl5b that was targeted to the neurites of hippocampal neurons was examined. In dissociated hippocampal neurons 21 DIV Arl5b was not diff usely distributed, but accumulated in aggregates (fi gure 3.5). However, it did not

Arl5b-GFP TGN46 GM130

Figure 3.3. Arl5b resides in the Golgi network in the soma of hippocampal neurons. Dissociated neurons were culti vated for four days and transfected with Arl5b-GFP. One day aft er transfecti on cultures were counterstained for TNG46, a trans-Golgi network resident protein, and GM130, a protein localized in the cis-Golgi. The white box in the overview (left ) indicates the magnifi ed region (right). Arl5b staining is enriched in the cis- and trans-Golgi. White bar in the overview indicates 20 μm and 5 μm in the magnifi cati on.

Additi onally, Arl5b was found in small, vesicular like structures at the leading edge of the growth cone of the developing axon (red arrows, fi gure 3.6). These vesicular like Arl5b aggregates were also en-riched in the shaft of the growth cone (blue arrow, fi gure 3.6) and along neurites (yellow arrows, fi gure 3.6).

Arl5b-GFP TGN46 Arl5b-GFP GM130

Arl5b-GFP TGN46 GM130 Merge

GM130 Arl5b-tdTomato Map2

Figure 3.4. Arl5b is not specifi cally enriched in the trans- or cis-Golgi network. For an improved analysis of Arl5bs distributi on in the Golgi apparatus dissociated hippocampal neurons were culti vated and trans-fected as indicated in fi gure 3.3.

Before counterstaining with TGN46 and GM130 neurons were incubated with 5μg/ml Nocodazole for 2 h for Golgi dispersion. Subsequent co-localizati on analysis of Arl5b either with TGN46 or GM130 confi rms overlapping signals with both mark-ers. Co-localizati on is labeled in white in overlay analysis of Arl5b/

TGN46 and Arl5b/GM130. The scale bar indicates 5 μm.

Figure 3.5. Arl5b is recruited to vesicular like structures in dendrites. Dissociated hippocampal neurons were transfected with Arl5b-tdTomato and culti vated unti l 21 DIV. Dendrites were visualized by immunostaining with MAP2 (blue) and somati c GM130 (green) highlights the Golgi apparatus. In the apical dendrite Arl5b (red) accumulates in vesicular structures. The white box in the overview (left ) indicates the magnifi ed dendrite (right). White bar in the overview indicates 20 μm and 5 μm in the magnifi cati on.

With a spinning disk live cell imaging setup the Arl5b positi ve vesicular like structures were tracked over ti me. These previously observed structures displayed a highly dynamic behavior during live cell imag-ing. For example aggregati on of Arl5b at the ti p of a fi lopodium was recorded. The formed Arl5b positi ve structure subsequently was targeted out of the fi lopodium and traffi cked along the neurite (fi gure 3.7).

Aft er their formati on most of the mobile vesicular like structures moved retrogradly along the de-veloping axon unti l they reached the soma of the neuron. This was observed in more than three independ-ent experimindepend-ents and in over 20 neurons in total. An example for the predominantly retrograde traffi cking

Arl5b-eGFP

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Arl5b-tdTomato

Figure 3.6. Arl5b aggregates in the growth cones and along neurites of dis-sociated hippocampal neurons. Confocal microscopy image of Arl5b-eGFP trans-fected cultured hippocampal neurons at DIV5. The depicted neuron was imaged 24 h aft er transfecti on. The white box in the overview (left ) marks the enlarged growth cone shown to the right. Red ar-rows mark accumulati on of Arl5b at the leading edge of a growth cone, the blue arrow points out an accumulati on of vesi-cle like aggregates of Ar5lb in the shaft of the growth cone and the yellow arrows show vesicular structures of Arl5b distrib-uted along the neurite. White bar in the overview indicates 20 μm.

Figure 3.7. Representati ve live imaging ti me series showing accumulati on of Arl5b followed by transportati on of the aggregate out of the fi lopodium. Spinning disk live microscopy reveals dynamic relocati on of Arl5b enriched puncta in neurites of cultured hippocampal neurons (transfected at DIV4 with Arl5b-tdTomato). Cells were recorded at DIV5 with 0.2 fps for a total of 20 min-utes. The sequence visualizes how Arl5b is enriched at a vesicle like structure in a fi lopodia (enlarged images) and is transported out of the fi lopodium into the neurite and towards the soma over ti me during the indicated ti me course. The scale bar indicates 10 μm.

3.1.3 Recruitment of Arl5b to membranes is GTP-dependent and requires N-terminal myristoylati on