Figure 3.5: Dynamics of actin clusters within CDRs. A - C: Time-lapse sequence of actin dynamics within CDRs. NIH 3T3 X2 cell, no stimulation, f-actin was stained with pLifeActGFP-TagGFP2. D: Overlay of the average intensity of the2.5 min-interval (green) and the standard deviation of the high-pass filtered sequence (red, cuto↵period: 1 min). The static cytoskeleton appears green, while red colors highlight dynamic structures within CDRs. Note that the wavefront is stationary. E: Close-up view of the red ROI inA(enhanced contrast), highlighting the growth front of an actin cluster (red solid line). F: kymograph sampled along the red dashed line in E with highlighted slope (dashed red line). G: Velocity distribution of actin clusters.
Scale bar in A:10µm.
Kymographs of individual HAPs also permit to read o↵ their typical lifetimes, which are typically below 30 s (Figure 3.5F). At fixed positions, the turnover from local high to low concentrations is on the order of 10 s, indicating high rates of polymerization and depolymerization.
Formation of contrast in all three techniques of microscopy that were applied so far can result both from di↵erences in local protein densities and from di↵erences in local cell thickness. For a more detailed investigation of the structure of CDRs and, especially, the actin distribution within them, three-dimensional imaging must be applied.
3.2 Three-Dimensional Structure and Morphology
While several aspects of CDR morphology and dynamics can be understood via a simplified picture, in which they constitute protein waves in a two-dimensional medium, for a deeper comprehension their three-dimensional nature must be taken into account.
We will see throughout this section that CDRs can form protrusions of considerable
46 CHAPTER 3. STRUCTURE AND MORPHOLOGY OF CDRS extension into vertical direction. A central question, which follows from this observation, is how these structures can translocate upon CDR propagation. Therefore, in this section the results of di↵erent approaches for a characterization of CDR in three dimensions are presented.
SEM micrographs from the literature on CDRs clearly show that they can consid-erably deform the cell membrane in vertical direction (Figure 1.1C) [Mellstr¨om et al., 1983, Dowrick et al., 1993, Edgar and Bennett, 1997]. This raises the question whether the vertical protrusions result from an up-piling of actin and the mere fact that the ver-tical direction is the only one that permits the formation of a protrusion. Alternatively, there could be a mechanism at work that restricts the polymerization of actin in CDRs to the dorsal membrane via an actin-recruiting protein that is membrane-bound, such as the proteins of the WASP family (Section 2.1.3).
To answer this question, cells exhibiting CDRs were simultaneously imaged in TIRF-and epifluorescence microscopy TIRF-and the resulting micrographs were then juxtaposed (Figure 3.6). In TIRF microscopy, only the first 100-200 nm-thick layer above the sub-stratum is illuminated while fluorophores situated further upwards remain dark [Murphy and Davidson, 2013, 252 pp].
Figure 3.6: Location of CDRs in vertical dimension. Upper row: epifluorescence, lower row:
TIRF. The cells in each column are identical. NIH 3T3 WT cells were stimulated with PDGF, fixed and stained with Rhodamin/Phalloidin. CDRs (marked with red arrows) are only visible in epifluorescence. Scale bars: 25µm
A comparison of images obtained via TIRF and epifluorescence reveals that CDRs are virtually invisible in TIRF micrographs. CDRs do, nevertheless, leave a footprint in the part of the cell that is close to the substratum in form of the aforementioned actin depletion within the area that is surrounded by their wavefronts. The CDR-forming
3.2. THREE-DIMENSIONAL STRUCTURE AND MORPHOLOGY 47 f-actin, however, is apparently completely situated at the dorsal side of the cell. This means that the reason for the formation of vertical protrusions of CDRs is not due to an up-piling of f-actin. Indeed, the results are a very strong hint towards the alternative hypothesis, i.e., an involvement of a membrane-bound protein in the recruitment of f-actin into CDRs. This membrane-bound protein would thereby only be found in the dorsal cell membrane. The identity of the latter will be further discussed in the final section of this chapter (Section 3.3).
To study the three-dimensional distribution of actin within cells forming CDRs further, Laser Scanning confocal Microscopy (LSM) was utilized. While TIRF is of outstanding resolution in z-direction it is limited to a single imaging plane, which is the layer directly above the substratum [Murphy and Davidson, 2013, 252 pp]. For LSMs the opposite is true [Murphy and Davidson, 2013, 265 pp]. While the resolution inz-direction of an LSM cannot compete with that of TIRF, the former can capture the entire vertical extension of a cell.
Figure 3.7 shows a cell exhibiting a CDR that was imaged via LSM. The corres-ponding image slices, in which image intensity is plotted as a function of heightz and a local spatial direction (s-direction), reveal the actin distribution within CDRs and the height of these structures. In comparison to images of epifluorescence and TIRF (Figure 3.6), where the presence or absence of CDRs is clearly visible to the human eye due to its capabilities of feature detection, the interpretation of xz-cuts of LSM images requires more dedication and a constant consultation of both, the slice images themselves (Figure 3.7b1-b4) and the maximum-intensity projection version of the whole stack (Figure 3.7B). A comparison of the cuts and the maximum intensity projection reveals that CDRs take ridge-morphologies, i.e., they consist of a ring-shaped bulge.
The magnitude of this protrusion, i.e., the CDR height, lies between 1 - 3µm in the example shown in Figure 3.7. An inspection of the actin distribution inside of the ridge reveals the consistency of the results from TIRF and LSM. The concentration of actin in CDRs is maximal within the part of the cell that forms the actual protrusion, while below these, i.e., towards the ventral cell side, we generally find lower concentrations of f-actin. Indeed, there are cases in which actin also appears in high concentrations below CDRs. However, a careful comparison of the slice cuts with the maximum-intensity projection (Figure 3.7B) and the focal plane of stress fibres (Figure 3.7A) reveals that this actin can be accounted to actin stress fibres that run below the CDR. Figure 3.7C
&D illustrate this based on an example of enlarged versions of the respective image parts. In general the amount of f-actin, and correspondingly the number of stress fibres, within CDRs is low compared to the remaining part of the cell, as can clearly be seen in Figures 3.7B and 3.7b1-b4.
Cells utilize f-actin for the formation of various di↵erent structures, some of them of relatively static nature such as, e.g., the actin cytoskeleton or the actin cortex. While it is apparent from Figure 3.7 that actin cortex and stress fibres contribute a level of
”background f-actin”, e.g. f-actin that is not involved in dynamic structures such as lamellipodia or CDRs, the same figure also reveals that above the cell nucleus only very
48 CHAPTER 3. STRUCTURE AND MORPHOLOGY OF CDRS little f-actin is found. This observation o↵ers an explanation to the avoidance of the nucleus exhibited by CDRs. Together, the observation of f-actin depletion inside of CDRs and the avoidance of the cell nucleus as a potential consequence of low f-actin concentrations above the nucleus, suggest that CDRs are built from actin that is, at least partially, recruited from the dissolution of actin stress fibres.
Despite its ability to image three-dimensional distributions of actin, LSM imaging goes along with some disadvantages. Besides the limits in resolution mentioned above, LSM imaging of CDRs requires the fixation of cells. The reason for this is that the time for acquisition of images such as, e.g., Figure 3.7 typically lasts several ten minutes, which is long enough to exceed the lifetime of a large proportion of CDRs. Fixation, in turn, involves the risk of creation of artefacts in cell morphology. Therefore, I validated my results with DIC microscopy, which is a label-free technique that can acquire images rapidly and thus enables live cell imaging.
The point spread function of a microscope operated in DIC mode quickly decays in z-direction, which makes it very suitable for optical sectioning [Allen et al., 1969, Kam, 1998]. While DIC cannot visualize protein distributions specifically, the principle of image formation of DIC makes it especially well-suited for the visualization of edges of objects. CDRs were therefore imaged on living cells with DIC optical sectioning to address the question of their three-dimensional morphology. I was especially interested in the clarification of a controversy in the literature, i.e., that in some articles CDRs are described as ”ridge[s] of extended dorsal plasma membrane”, i.e., flat membrane structures, containing ”numerous similarly-sized bumps,” [Buccione et al., 2004]. In contrast, other publications emphasize their sheet-like morphology [Mellstr¨om et al., 1983,Dowrick et al., 1993], or di↵erentiate between flat and sheet-like ru✏es in assigning them to di↵erent types [Edgar and Bennett, 1997].
The microscope was set up to acquire image stacks of living cells at equally spaced z-positions. The time between individual images was on the order of tenth of seconds, i.e., CDRs could not alter their shape or position significantly within acquisition of a completez-stack.
The subset of images of a z-stack shown in Figure 3.8 presents a CDR that exhibits both, a ridge-shaped region resembling a flat and smooth bump, while in other parts
Figure 3.7 (facing page): CDR imaged with LSM.F-actin: yellow (Rhodamin/Phalloidin), DNA: blue (DAPI).A: Ortho-slice visualization of a 3-d image stack (blue: xy-, green: xz-, red: yz-plane). The position of the CDR is highlighted with white arrowheads in thexz-view.
The lower cell part is in focus in the xy-view and the CDR thus only appears very weakly. B:
Maximum-intensity projection of the same image stack. White lines indicate the positions where the slice cutsb1-b4 where taken. The dashed white lines inb1-b4 highlight thez-position of the substratum. The origin of the local directionslies outside of the cell. C: enlarged ROI from Aaround slice cutb1 with numbered stress fibres. D: slice cut b1 in which the same stress fibres are highlighted with white circles. NIH 3T3 WT cell, PDGF stimulation, fixation, scale bar: 25µm