Effect of cytoskeleton perturbations and modulation of ion transport activity
Cytoskeleton rearrangements play a major role in cell spreading: actin polymerization drives spreading and myosin activity resist to it (Wakatsuki 2003). For example, we observed homogeneous actin distribution for suspended cells and during spreading actin was accumulating close to the contact area (Fig. 1.7). Thus, to further check our hypothesis that the speed of spreading deformation regulate volume loss while spreading, we decided to modulate the speed of spreading on fibronectin-coated glass by acto-myosin cytoskeleton perturbations: actin depolymerization, inhibition of Arp2/3, inhibition of contractility. Figure 1.7 LifeAct distribution (black) in spreading cell at different time points, side-view. Before spreading (time zero), actin is distributed homegenously on the cell surface. After initial spreading, actine accumulates at the contact area (10 min).
Inhibition of actin polymerization
Although the early stage of spreading was shown to be passive (Cuvelier et al. 2007), later stages of spreading, including lamellipodia formation, require actin polymerization. Therefore, we decided to check the effect of actin disruption by Latrunculin A (Lat) treatment on average volume dynamics during spreading. Cells treated with a high dose of Lat (2 µM) were blebbing without spreading and, similarly to cells platted on PEG substrates (non-adhesive), did not exhibit loss of volume, but rather an increase, in the first 30min after platting (Fig. 1.8a). On longer timescales, treated cells lost volume, without spreading, which could be due to the known long term effect of Lat on cell growth(Spector et al. 1989).
Cells treated with a low dose of Lat (100 nM) were also blebbing, and spread at a slower speed than control cells. They lost only 2% of volume on average (Fig. 1.8a,b). Analysis of individual trajectories showed that 27% of cells did not lose volume while spreading (more than control cells, 15%). Further analysis showed that Lat treated cells tend to rather increase their surface area than decrease volume as they spread (Fig. 1.8c). Analysis of and the average spreading area showed that Lat treatment slowed down spreading (Fig. 1.8b,e), if compare with control cells. Volume flux (Fig. 1.8f) was proportional to the speed of spreading and similar to control cells that were spreading within a low range of speed (Fig. 1.8d), confirming that depended on .
(a): Average volume of control cells (blue), treated with Lat 100nM (yellow) and 2µM (gray) of a typical experiment. In the first 20 min of experiment cells treated with 2 µM Lat did not spread and did not lose volume; cells treated with 100 nM Lat lost less volume than control. Error bars represent standard error.
(b): Average spreading area of control and Lat 100 nM treated cells of a typical experiment. A low dose of Lat delays spreading. Error bars represent standard error.
(c): Volume-surface balance for control and 100 nM treated cells.
(d): Median values of for the different intervals. Error bars represent standard deviation.
Inhibition of Arp2/3
To obtain a more specific perturbation of actin dynamics, we chose to affect Arp2/3, the nucleator of branched actin networks, responsible for lamellipodial formation and thus important for fast cell spreading. Cells treated with CK-666 (100 µM) lost less volume than control cells on average (~2%) and had a smaller spreading area (Fig. 1.9a,b, g). CK-666 treatment reduced volume loss by reducing both and in the similar manner than treatment with low dose of Lat: volume flux remained proportional to the spreading speed (Fig. 1.9d-f).
The slowing down of spreading by CK-666 and Lat is consistent with previous research and could be explained by inhibition of actin polymerization and contractility increase (Q. Yang et al. 2012; Bun et al. 2018) and assembly of a different actin architecture (Henson et al. 2015), less favorable to fast spreading.
Modulation of fast spreading without volume loss
Our results so far showed that we can modulate volume loss associated to cell spreading by affecting the cell cytoskeleton, with the general rule that faster spreading cells tend to lose more volume. We wondered if it is possible to break this rule, for example having fast spreading cells that do not lose volume while spreading. To achieve this, we reasoned that on one hand, reducing contractility increases spreading speed by making cells softer, but also that it is known to indirectly activate cell protrusion activity due to mutual negative interactions between the RhoA (contractility) and Rac1 (protrusion) pathways. We thus examined the effect of inhibition of Arp2/3 in cells with low contractility by a combination of CK-666 and Y-27 treatment, which does not affect cell viability at all contrary to other combinations of drugs. Double treated cells spread fast, like cells treated only with Y-27 (Fig. 1.12b, g), but they did it without volume loss, just like cells treated only with CK-666: double treated cells lost 2% of volume on average, and 25% of cells did not lose volume while spreading (Fig. 1.12a). At the early stage of spreading, cells increased surface area rather than losing volume (Fig. 1.12c), even if they were spreading fast. Distribution of was similar to Y-27 treated cells (Fig. 1.12e), however, distribution of was shifted towards even positive values (Fig. 1.12f). This result shows that fast spreading can occur without volume loss (Fig. 1.12d). It allows us to start drawing a working model consistent with this ensemble of results (see next chapter for a more detailed version of the model): reducing contractility allows fast spreading, which, in cells treated with Y-27 only, is accompanied with increased lamellipodial expansion. This would lead to fast and large volume decrease because inhibition of contractility on one hand can prevent membrane tension relaxation by bleb formation and on the other hand can increase tension due to fast pulling, from the cell cortex, of the membrane area needed to form the extending lamellipodium. Additional inhibition of branched actin polymerization would then prevent volume loss, either because it prevents membrane stretching by expansion of the thin lamellipodial protrusion produced by branched actin. Alternatively Arp2/3 inhibition rescues blebbing or any other sort of membrane detachment from the underlying cortex, which would help relaxing membrane tension during spreading. This could be because branched actin produces more friction on the plasma membrane, and thus more tension when it expands/unfolds during cell spreading; or because branched actin is required to activate ion transport (Chifflet and Hernández 2012) and consequent volume loss; or because it binds to particular membrane associated structures associated to ion pumps and involved in cell volume modulation (e.g. caveolae (Balijepalli and Kamp 2008)).
Volume modulation during spreading of RPE-1 cells.
To extend our finding to other cell types, we studied spreading of RPE-1 cells, which is known to have a lower basal contractility than HeLa cells (Liu et al. 2015). We first established the same basic volume modulation behavior during spreading (Fig. 1.15a,b,c). Cells placed on PLL-PEG did not spread and did not exhibit volume loss, instead, volume was increasing with the speed similar to cell growth, ~3.5%/hr. Cells plated on fibronectin displayed, in the initial phase of spreading, an almost opposite behavior to HeLa cells: during the first 10 min they increased their volume by 3% on average. After this brief phase of increase, the volume decreased by about the same extent and at the same speed as in HeLa cells, reaching about 95% of initial volume in the following 20 minutes (Fig.1.15b). Importantly, the phase of volume loss actually corresponded to the phase of lamellipodial extension (see IRM images in Fig. 1.15a, 10 first minutes are just adhesion of the cell body and after 10 minutes lamellipodia form). Analysis of individual trajectories showed that, indeed, a majority of cells (82%) increased volume at the early stage of spreading and then volume decrease corresponded to lamellipodia formation (confirming our previous observations). Accordingly, cells tended to extend surface rather than lose volume at the early stage of spreading (Fig.1.15d).
Table of contents :
1. What is cell made of?
a. Plasma membrane
d. Cytoplasm and nucleus
2. Methods to study the mechanical properties of a single cell
a. Atomic force microscopy (AFM)
b. Micropipette aspiration
c. Tether pulling
d. Particle tracking
3. Cell volume regulation in response to deformations of different timescales
a. Steady-state volume
b. Osmotic shock
1. Cell culture and drug treatment
2. Measurements of ATP depletion effect on cell viability
3. Monitoring of cell volume and contact area while spreading
a. Chambers and cell preparation
c. Raw data extraction
d. Computation of apparent surface area
e. Computation of spreading speed and volume flux
4. Cell volume measurements under confinement
a. Static 6-well confiner
b. Dynamic confiner
6. Cell volume measurements of dendritic cells (DCs) in collagen gel
7. Cell volume measurements during osmotic shock
8. Side-view microscopy
9. Spinning disk microscopy
10. Mass measurements
1. Dynamic cell spreading
a. HeLa cells lose 5% of volume on average while spreading
b. Diversity in single cell behavior. Fast spreading induces fast volume loss
c. Effect of cytoskeleton perturbations and modulation of ion transport activity
d. Summary of results on spreading HeLa cells and working model
e. Volume modulation during spreading of RPE-1 cells.
f. Na+/H+ exchanger (NHE1) inhibition
g. General conclusion on analysis of single cell spreading experiments
2. Cell population analysis of the coupling between shape and volume
a. The volume of a cell is independent of the size and shape of its spreading area
b. Conclusion on steady state spread cells
3. Volume modulation during imposed deformation by mechanical confinement
a. Cell shape imposed by 2D confinement
b. Confinement induces volume loss
c. Actin disruption prevents volume loss under confinement
d. Confinement induces volume loss in tens of milliseconds
e. Cells do not lose dry mass upon confinement
f. Confinement induces death of ATP depleted cells, but not of control cells
g. Other cell lines
4. Osmotic shocks
a. Estimation of total plasma membrane area
b. Initial passive response to osmotic shocks at the level of the cell population
c. Diversity of passive response at the level of individual cells
d. Dynamics of the passive response to hypoosmotic shock
e. Regulatory volume decrease/increase
IV. Discussion and perspectives
1. Osmotic shock