3.8. A system to test DAAM1 involvement in cell migration
3.8.1. DAAM1 is essential for Golgi apparatus orientation in cell
In this cell type, the percentage of cells showing Golgi orientation one hour after scratch was 70-80% compared with the starting point of 30-40% which is as good a range as for other cell types, but over a considerably shorter time. In DAAM1 depleted cells, the forward orientation remained at 30-40% for siRNA si2318 and 50- 60% for the less effective si2832 which correlated with protein depletion. In order to confirm that the pathway involving Golgi re-orientation is conserved in COS 7 cells, various control inhibitors were tested for their effects on orientation and wound
Chapter 3: Results
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Figure 3.23 Migration of COS 7 in wound healing assays
(a) Top: siRNA knockdown of DAAM1 in COS 7. Bottom: Time taken for wound closure did not differ between cells transfected with control or DAAM1 siRNA.
Cells were transfected with control (Ctr), si2318 or si2832 siRNA. One day after transfection, the media was replaced with 1% serum-containing media in the evening and cells were incubated overnight. A scratch was made the next day using a yellow pipette tip across the monolayer. Cells were imaged under the Zeiss Axiovert 135M Inverted microscope equipped with a Zeiss 10x/0.25 Plan-APOCHROMAT lens
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Figure 3.23 Migration of COS 7 in wound healing assays
(b) COS 7 cells transfected with DAAM1 siRNA (si2318) were wounded and imaged under the Zeiss Axiovert 135M Inverted microscope equipped with a Zeiss 10x/0.25 Plan-APROCHROMAT lens to record the movement of cells upon serum replacement after wounding. Images were enlarged to show the presence of random protrusions in wound edge cells.
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Chapter 3: Results
Figure 3.24 Golgi re-orientation of COS 7 in wound healing assays
Top: A cell was scored positive if at least 50% of its Golgi apparatus falls within the front 120º sector. Bottom: Percentage of Golgi re-orientation in wounded COS 7 cells transfected with control or DAAM1 siRNA. Cells were transfected with control (Ctr), si2318 or si2832 siRNA. One day after transfection, the media was replaced with 1%
serum-containing media in the evening and cells were incubated overnight. A scratch was made the next day using a yellow pipette tip across the monolayer. Cells were allowed to recover for 15 minutes before being fixed for the 0 time-point. For the 1 hour time-point, 10% serum-containing media was replenished after the recovery period and cells were fixed an hour later. Results were based on 3 independent experiments. At least 100 cells were counted for each set of experiment.
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healing in COS 7 cells. Both PKC and GSK inhibitors inhibited Golgi orientation to some degree (Figure 3.25). Surprisingly, blebbistatin, an inhibitor of myosin II, had no effect on Golgi orientation even though myosin II is required for nuclear movement and MTOC orientation in fibroblasts [95]. I attribute this to the lack of myosin IIA in this cell type [96]. The presence of myosin IIA fibres may prevent nuclear movement and/or MTOC orientation and therefore require remodelling in say mouse fibroblasts. This could be tested by determining if MRCK inhibition has any effect in this cell type [95]. Interestingly, methyl- -cyclodextrin (that depletes cholesterol) had no effect on Golgi orientation although it strongly inhibited cell migration. It might be that loss of lipid rafts blocks Rho signalling at the plasma membrane as suggested [61, 97] but does not affect the function of intracellularly located DAAM1. As expected, cytochalasin D which induces depolymerisation of F- actin inhibited Golgi orientation, underlining the importance actin fibre organization in the process of cell directionality and positioning.
In detailed examination of the DAAM1-depleted COS7 cells, DAAM1 siRNA transfected cells had leading edges that were more “branched” than for the control siRNA transfected monolayers (Figure 3.26) suggesting that the cells develop several
“leading edges”. This is consistent with the notion that there is a failure in cell polarity cues in the absence of DAAM1. In transient transfection assays, we were also able to show that PAK inhibition using the KID [98, 99] could block Golgi re- orientation as well as various dominant inhibitory versions of the GTPases (Figure 3.27). In my hands, the efficacy of dominant inhibitory versions were in the increasing order of Cdc42>Rac1>RhoA.
Chapter 3: Results
Figure 3.25 Effects of different drug treatments on Golgi re-orientation in COS 7 Top: Percentage of Golgi re-orientation 1 hour post-wounding in cells with different treatment. Results were based on 3 independent experiments performed for each treatment. At least 100 cells were counted for each set of experiment. PKC inhibitor RO-320432 20 àM, GSK inhibitor SB216763 20 àM, methyl- -cyclodextrin (M CD) 5 mM, blebbistatin 50 àM, cytochalasin D (CyD) 2àM. Bottom: Confluent monolayer of cells were treated with 5 mM M CD for an hour before a scratch was made. Cells were then imaged under the Zeiss Axiovert 135M Inverted microscope equipped with a Zeiss 10x/0.25 Plan-APROCHROMAT lens to record the movement of cells upon serum replacement after wounding.
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Figure 3.26 Tubulin staining of COS 7 wound edge cells
Cells were transfected with DAAM1 siRNA or treated with the respective drug for 1 hour before wounding. PKC inhibitor RO-320432 20 àM, GSK inhibitor SB216763 20 àM. Cells were fixed 1 hour after replacement of 10% serum-containing media and stained using anti- -tubulin. The wound is located at the bottom of each panel (arrow).
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Figure 3.27 Effects of αPAK KID and dominant inhibitory Rho GTPases on Golgi re-orientation in COS 7 cells
Cells were transfected with control vectors or plasmids expressing αPAK kinase inhibitory domain (KID), Rho N19, RacN17 or Cdc42N17 before wounding.
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Chapter 3: Results
When the cells were subjected to live-imaging, I was able to directly visualise the Golgi either by using the GFP-tagged Mannosidase II or by direct observation of the DIC movies, since the Golgi is well defined as an asymmetric bulge on the side of the prominent nucleus. Unlike the process reported in fibroblasts [95], it appears that the re-orientation of the Golgi was mediated by a rotation of the nucleus (Figure 3.28) as assessed by the positions of the nucleoli, rather than a disconnection and movement of the nucleus from the Golgi apparatus. The similarities and differences between cell types in the process of Golgi orientation reflect the complex processes involved and indeed in some systems the MTOC/Golgi has been observed to orientate away from the direction of movement [100]. Nonetheless, these observations are the first to implicate a formin in this process that potentially links Wnt signalling to events in the Golgi including the directional secretion of proteins in development and cell movement.
Chapter 3: Results
Figure 3.28 Golgi re-orientation and nuclear rotation in COS 7
(a) Confocal imaging of control siRNA-transfected COS 7 cells using Olympus Fluoview FV1000-MPE microscope with a 60x/1.45 Plan-APOCHROMAT lens.
Cells were transfected with Mannosidase II-GFP to mark the Golgi. Positions of nucleoli were marked to indicate relative position of nucleus to the Golgi.
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Figure 3.28 Golgi re-orientation and nuclear rotation in COS 7
(b) Confocal imaging of DAAM1 siRNA-transfected COS 7 cells using Olympus Fluoview FV1000-MPE microscope with a 60x/1.45 Plan-APOCHROMAT lens.
Cells were transfected with Mannosidase II-GFP to mark the Golgi. Positions of nucleoli were marked to indicate relative position of nucleus to the Golgi.
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