Cytoskeletal functions are not compromised by expression

The findings in the previous chapters demonstrated that Lifeact could indeed be used to visualize the actin cytoskeleton in mammalian cells. I now wanted to test, whether Lifeact expression leads to measurable changes in actin dynamics in transfected cells. To this end, I studied three parameters that are dependent on a functional actin cytoskeleton: cell polarization, retrograde actin flow in lamellipodia and directed cell migration.

Neuronal polarization is essential for development and functionality of these cells. To determine and establish one axon out of all dendrites is strongly dependent on a dynamic actin cytoskeleton (Witte & Bradke 2008). To examine possible neuronal polarization defects, primary neurons were prepared from rat hippocampi, transfected

with either Lifeact-EGFP or EGFP-actin and cultured for three days before fixation and staining for Tau-1. This protein is only present in the mature axon and therefore was used as characteristical marker. These two samples as well as mock transfected control cells were subsequently analyzed for their polarization stage. Cells were categorized as either having no axon, one axon or more than one axon. I found that neuronal polarization was not significantly affected by the expression of Lifeact-EGFP (Fig. 3.12, 60.1±0.2 % cells formed one axon compared to 68.5±8.7 % of mock transfected cells) but a comparable expression of EGFP-actin led to a significant alteration in the polarization stages (52.3±4.4 %, ANOVA: F2,8 = 6.205, P < 0.0346; post-hoc Dunnett's test: P > 0.05 for Lifeact-EGFP, P < 0.05 for EGFP-actin). Hence, these results indicated that Lifeact expression does not affect neuronal polarization. In contrast, EGFP-actin expression led to impairments in performing this process.

Lifeact-EGFP EGFP-actin

control

Figure 3.12 Quantification of neuronal polarization. Primary rat hippocampal neurons were transfected with either Lifeact-EGFP or EGFP-actin and cultured for three days. Then, they were analyzed for the presence of axons in comparison to mock transfected cells. Data shown are averages ± SD from at least three experiments.

I next measured the speed of retrograde actin flow in lamellipodia of MEFs. In lamellipodia actin forms a highly dynamic network of branched filaments (Chhabra & Higgs 2007). During treadmilling, these filaments move from the cell periphery in direction to the cell body and this movement is called retrograde flow. The speed of

this process is dependent on the actin kinetics in vivo and hence, slight changes can directly be observed by its measurement (Lin & Forscher 1995, Medeiros et al. 2006). To this end, I transfected MEFs with either Lifeact-EGFP or EGFP-actin and imaged the cells with TIRFM. As a control, I used untransfected cells which were imaged by differential interference contrast (DIC). Using this method, I monitored the retrograde membrane flow which corresponds to the retrograde actin flow in cells and thus, provided a tool for measuring the speed in non-manipulated cells. The analysis of the speed of retrograde flow in lamellipodia revealed that Lifeact-EGFP transfected fibroblasts were indistinguishable from non-transfected cells at 4 µm/min whereas the retrograde flow was reduced to about half in EGFP-actin expressing cells (Fig. 3.13; ANOVA: F2,134 = 53.39, P < 0.0001; post-hoc Dunnett's test: P > 0.05 for Lifeact- EGFP, P < 0.05 for EGFP-actin). These results supported the previous finding that EGFP-actin disturbs actin kinetics and clearly demonstrated that Lifeact does not interfere with retrograde actin flow.

***

*

Lifeact-EGFP EGFP-actin control

Figure 3.12 Quantification of the speed of lamellipodial retrograde actin flow. MEFs were transfected with either Lifeact-EGFP or EGFP-actin and imaged by TIRFM. Untreated control cells were imaged by DIC. The velocity of the retrograde flow was measured from kymograph traces. P > 0.05 (*) for Lifeact-GFP, P < 0.05 (***) for EGFP-actin. Data shown are averages +/- SD from at least three experiments.

Finally, I examined the chemotactic speed of dendritic cells. These cells are mediators of the adaptive immune response while presenting antigens to lymphocytes. They recognize invaders in the periphery of an organism, e.g. the skin. After engulfing the

invaded particles, they migrate towards the draining lymph node, along a chemokine gradient (CCL19 or CCL21), where they activate lymphocytes (Montoya et al. 2002). First, bone marrow precursors were matured to dendritic cells and transfected with either Lifeact-EGFP or EGFP-actin. After sorting positive cells by FACS, they were embedded in a three-dimensional collagen-matrix (Lammermann et al. 2008). Video- microscopy was performed to follow migration of these cells towards a chemokine (CCL19; Figure 3.13a). The speed of single cells was compared to non-transfected control cells. Lifeact-EGFP expression had no significant effect on the speed of chemotactic dendritic cell migration (paired t-test (two-tailed), P = 0.40, n = 3 experiments, 837 tracked cells), while EGFP-actin expressing cells migrated slower than control cells (P = 0.04, n = 4 experiments, 689 tracked cells) (Figure 3.13b).

CCL19 solution

a

b

Figure 3.13 Comparison of chemotactic speed of dendritic cells. a) Schematic drawing of experimental setup: primary dendritic cells were embedded in a three-dimensional collagen gel and a solution of the chemokine CCL19 was applied to the top of the matrix. Migration was monitored by video-microscopy and migrating cells were tracked using Metamorph software (Molecular devices). b) The chemotactic speed of transiently transfected dendritic cells relative to untransfected cells is shown. P = 0.40 (*) for Lifeact-EGFP, P = 0.04 (***) for EGFP- actin. Data are averages ± SD from at least three experiments.

Taken together, expression of Lifeact in mammalian cells did not lead to significant changes in actin dynamics during neuronal polarization, lamellipodial retrograde actin flow and speed of chemotactic dendritic cells. However, EGFP-actin expression significantly altered actin dynamics in these processes.