Formation and Cooperative Behaviour of Protein Complexes on the Cell Membrane (Springer Theses)

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It seems that you're in Germany. We have a dedicated site for Germany. Its theme can be described as the classical Rutherford scattering experiment adapted to the LHC: measurement of scattering angles to search for new physics and substructure. At the LHC, colliding quarks and gluons exit the proton collisions as collimated particle showers, or jets. The thesis presents studies of the scattering angles of these jets. It includes a phenomenological study at the LHC design energy of 14 TeV, where a model of so-called large extra dimensions is used as a benchmark process for the sensitivity to new physics.

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Membrane shape-mediated wave propagation of cortical protein dynamics | Nature Communications

Simone Napolitano. As the wave propagates, the wavefront marks the highest F-BAR intensity and hence membrane curvature. In contrast, the membrane height continues to increase in the trailing path behind the F-BAR wavefront. We note that while SRIC is sufficient to detect significant membrane-substrate distance changes, its spatial resolution is too limited to precisely map out actual membrane curvature at finer scale Nevertheless, our findings indicate a strong correlation between local membrane shape change and rhythmic propagation.

Furthermore, this observation demonstrates that F-BAR dynamics synchronizes with membrane shape deformation, suggesting curvature sensing is at play, consistent with our model proposal. As membrane shape did change detectably along the wave propagation path, we determined whether membrane shape changes were necessary for wave propagation. The model predicts that wave propagation has specific requirements on the curvature sensitivity and recruitment rate of F-BAR Fig.

To test this prediction, we first examined the effect of removing F-BAR proteins on the formation of traveling waves. We reduced the level of endogenous curvature-generating proteins by shRNA. This indicates some redundancies among the Toca family of F-BAR proteins and demonstrates their essential role as a collective entity of the Cdc42 interacting F-BAR proteins in the traveling waves.

Wave propagation requires F-BAR and its curvature sensitivity. The red dot represents the model parameter set that generated the nominal model results in Figs. Error bars: s. Gray area indicates the curvature range that allows wave formation, corresponding to the gray area of a. Mutants are separated into four groups based on curvature-generating abilities. Because F-BAR proteins could function in regulating actin dynamics in addition to their membrane-remodeling ability, the indispensable role of F-BAR in wave propagation did not necessarily prove a requirement for membrane shape changes.

To further explore whether the wave propagation requires changes in membrane shape, we generated a series of domain-swapping mutant proteins that differed from FBP17 only in their curvature preferences and tested whether they could functionally replace FBP17 in the waves. Phospholipid-binding but curvature-insensitive PH domain of phospholipase C 46 or membrane-targeting sequence of tyrosine-protein kinase Lyn Lyn10 were introduced as controls. Among these, the mutant with the constitutively membrane-targeting Lyn10 did not form waves in wild-type WT cells, consistent with a requirement of F-BAR dissociation from the membrane for waves Fig.

The rest of the domain-swapped mutants still localized to the waves in WT cells, indicating that these mutants were properly folded functional proteins. The fact that the BAR from Endophilin2 or ENTH from Epsin1 domain-swapped mutant could not rescue wave formation suggests that a specific range of curvature similar to or lower than that of the F-BAR domains is required for wave formation, as predicted Fig.

Of particular interests are mutants KA and K66E, which generated narrower higher curvature and wider tubules lower curvature compared with wild-type F-BAR domain, respectively, in in vitro liposome tubulation assays Together with the domain-swapping data Fig. We conclude that membrane shape-mediated feedback is actively involved in the cortical wave propagation.

The requirement of membrane shape-mediated feedback confirmed the mechanochemical nature of the wave propagation. An independent test for this was on the mechanosensitivity of the waves. As membrane deformability critically determined rhythmic propagation of cortical protein recruitment in our model, the wave propagation was predicted to diminish with increasing or decreasing membrane tension Fig.

Hence, we investigated the effect of membrane mechanics on the waves. We conducted osmolarity shock experiments by cyclically perfusing cells with hypotonic or hypertonic buffers; these treatments increased or decreased membrane tension, respectively, which subsequently affected membrane shape deformability. The kymographs show that osmolarity changes reversibly inhibited rhythmic wave propagation Fig.

Osmotic treatment could lead to additional effects, including cell volume and cytosolic concentration changes Supplementary Fig. To circumvent these potential side effects, we used the surfactant deoxycholate DC to specifically reduce the plasma membrane tension without changing cell volume Such effects were reversible on the time scale of seconds upon DC washout, eliminating possibilities of permanent membrane compositional changes. Waves experiencing moderate DC or hyper-osmotic treatment displayed no significant changes in propagation speed, but prolonged oscillation periods Fig.

Membrane mechanics is essential for wave propagation. The red dot represents the model parameter set that generated the nominal model results in Fig. Bottom left: fast Fourier transform FFT shows the dominant periods in each duration. Bottom right: wave speed distribution in each duration. Colors of plots indicate durations as in top. For b — d all the schematics above kymographs illustrate putative changes in cell membrane tension. So far, we demonstrated that membrane shape change and protein curvature sensitivity were integral to our mechanochemical wave.

While the membrane undulation wave is mechanical, the cortical protein traveling waves are chemical waves. Does this ultrafast wave speed depend on the membrane shape-mediated feedback? What is the nature of such protein waves? In conventional reaction-diffusion systems e. Here real wave is driven by diffusion—a real propagation of chemicals in physical space 1.

In contrast, pseudowave is not a real material propagation in space; instead, it reflects the spatial gradient in the timing of local excitation that gives the propagating impression. The question is: in the context of chemical traveling waves, is our cortical protein wave propagation more akin to real or pseudowave? To gain insight in cortical protein traveling waves, we carried out more vigorous mathematical analysis of our system. By ignoring protein lateral diffusion along the cortex, we simplified the model to make it possible to obtain analytical solution of traveling wave.

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A nontrivial steady-state traveling wave solution emerged with a nonzero membrane curvature at the wavefront. Interestingly, without protein lateral diffusion, this analytical solution yielded similar wave speed as those from the full model Fig. This finding suggests that diffusion need not be important in wave propagation, indicating that our protein traveling wave may not reflect real material movement in the direction of wave propagation. To further pinpoint the nature of our protein traveling wave, we borrowed the criteria that distinguishes pseudowave from real wave 49 : a wave is a pseudowave if the concentration changes—resulting from temporal chemical reaction at each point in space—are much larger in magnitude than those resulting from diffusion.

In this regard, diffusion in our nominal case contributed little but negatively to the wave propagation over time Fig. Taken together, we suggest that our cortical protein traveling wave is more in line with pseudowave. Importantly, this is the only type of cortical protein traveling wave in our system—a unique feature distinct from conventional reaction-diffusion systems—that typically host both real and pseudowaves.

Curvature sensing-mediated cortical protein waves always reflect protein recruitment from cytoplasm onto membrane. The analytic result was obtained by computing the analytical solution Eq. We note that there is one parameter in the analytic formula that cannot be analytically derived from the reaction rates in the full model.

And we chose the location ahead of the wavefront, where the absolute value of membrane curvature is the highest. Variation in this location does not change the qualitative conclusions from these plots. We emphasize that the comparison here is only within the regime of traveling waves, not stationary wave phenomena that could emerge with characteristic spatial periodicities from conventional reaction-diffusion systems.

The experimental data were collected from 37 waves in 8 cells. The F-BAR intensity for each wavefront was normalized to the maximum value within the individual cell. The gray shade represents the tracking measurement uncertainty Supplementary Figs. We then asked: what underlies this unique feature? While all traveling waves must have the promotion zone ahead of the wavefront to confer propagation Fig. In conventional reaction-diffusion systems, as lateral diffusion advances the wavefront, autocatalytic reactions of the local chemicals always promote excitation in the direction of wave propagation, because the chemical concentrations are always positive Fig.

In contrast, the stimulatory elements in our system include not only conventional chemical autocatalytic reactions but also curvature sensing effects. The membrane curvature changes sign from negative to positive as one moves away from the wavefront in the direction of wave propagation Fig. Because F-BAR only accumulates in the negative-curvature region, it is not recruited to the positive-curvature region due to the geometric mismatch. As such, the curvature sensing constrains the effect of protein diffusional drift on wave propagation Fig. We therefore suggest that curvature sensing-mediated traveling waves of cortical proteins mainly reflect the local protein recruitment from cytoplasm, rather than the protein lateral diffusion Fig.

What really propagates here is the membrane undulation that induces local protein recruitment from cytoplasm, potentiating wave propagation like a ripple moving in a pond. For the corresponding cortical protein traveling wave, it is the spatial gradient in the timing of local excitations that gives the propagating impression.

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Hence, the smaller this spatial gradient, the smaller the relative timing in excitations and, hence, the faster the protein wave appears to propagate. To test this prediction, we used TIRFM experiments to track wavefront propagation with high spatial-temporal accuracy. For a given wave, wave speed is heterogeneous. Importantly, our experiments show that the spatial gradient of cortical protein density at the wavefront inversely correlates with the wave speed circles, Fig.

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Because the spatial gradient of cortical proteins reflects the relative timing of the local excitation Fig. More critically, a zoom-in kymograph shows that the individual FBP17 punctum undergoes cycles of assembly and disassembly during wave propagation, but do not notably move in space Fig. We conclude that our cortical protein traveling waves mainly reflect the protein recruitment from cytoplasm, rather than, the lateral diffusional drift. In this work, we provide some mechanistic insights into cortical traveling waves.

Our work highlights the importance of membrane shape change in establishing a mechanical cue for cortical protein recruitment, which reciprocally governs cortical dynamics that culminate in traveling waves. Critically, we show that in a curvature sensing-driven rhythmic propagation, the cortical protein traveling wave results mainly from the local protein recruitment from the cytoplasm, rather from lateral diffusion.

Our cortical protein traveling waves have several unusual features. This is because the protein traveling waves emerge from the membrane shape-mediated feedback, and are constrained by the accompanying membrane undulation that itself is a real wave. Second, while the speed of our mechanochemical wave is ultrafast, it is still modulated by the protein lateral diffusion along cortex. This is different from another interesting example of a mechanochemical wave 53 , whose predicted wave speed is independent of protein lateral diffusion, as this chemical wave is entirely driven by and, hence, effectively reads out, membrane mechanics In contrast, in our model wave speed is controlled by the feedback between membrane curvature and cortical protein dynamics, and is not completely independent of protein lateral diffusion Fig.

Third, in our model the sub-diffusive dependence of wave speed on protein lateral diffusion constant is a consequence of curvature sensing, not just because of the mechanochemical feedback. To demonstrate this, we altered the model in several ways, maintaining mechanochemical feedback but without curvature sensing, and found that the corresponding wave speed was not always sub-diffusive Supplementary Fig. In contrast, the wave speed in the other model schemes—that preserve curvature sensing in the feedback—is always sub-diffusive Fig.

Interference between traveling waves could make it possible for cells to coordinate and integrate cortical signals. While our model focuses on a specific system, it provides a general framework for curvature sensing-mediated traveling wave dynamics. The unusual features remain robust to variations in the detailed model scheme.

They only require autocatalytic cortical recruitment of a curvature-sensitive protein coupled with a negative feedback possibly involving actin dynamics Supplementary Fig. Given that there are many other curvature-sensitive proteins involved in diverse cellular processes 5 , 18 , 54 , 55 , 56 , the biological implications of our model can be broad. We note that curvature sensitive I-BAR domain proteins e. Our model recapitulates the basic features of our traveling wave but is inevitably incomplete.

First, we focused on the cortex-centric mechanism of rhythmic propagation. An open question is: can the cortex-bound protein wave be part of a real wave in the cytoplasm? Even if the wave exists in the cytoplasm, we reason that the membrane shape change and the curvature sensitivity of F-BAR are still essential for this rhythmic propagation Figs. Future work will investigate the possibility of traveling waves with a cytosolic origin. Second, the model treated the ventral membrane at the cell edge as a clamped boundary.

While external stimulation i.

Formation and Cooperative Behaviour of Protein Complexes on the Cell Membrane

This is because the membrane at the epicenter does not entirely relax back to the baseline after the protein wave has passed. It is this residual membrane shape deformation that serves as a cue to recruit F-BAR, which in turn initiates the next round of oscillation Supplementary Fig. On the other hand, because the clamped boundary dampens the membrane shape changes, its effect propagates from the edge to the epicenter, where it flattens the residual membrane shape deformation over time. Without external activation signals, this flattening of the membrane eventually prevents a new round of F-BAR cortical recruitment and, hence, dampens the oscillation at long times.

This is consistent with our observations that the waves are only on the cell ventral side and not strictly sustained oscillations as indicated by Fig. Nevertheless, our key conclusion remains robust regardless whether the dynamics is a sustained or dampened oscillation. In reality, the cell edge may evolve over time; and membrane-substrate adhesion is probably not spatially uniform as assumed.

Interestingly, a localized adhesion that clamped the membrane diverted the wave propagation Supplementary Fig. In the future, we would like to systematically investigate how dynamics of cell edge movement and cell-substrate adhesions impact our traveling wave. Third, our model proposed that the membrane stiffening by actin accumulation prevented membrane deformation and hence turned off F-BAR recruitment.

This is consistent with a negative role of actin in cortical wave observed previously 3 , and supported by our observations that the actin accumulation negatively correlates with the membrane height change Supplementary Fig. Also, latrunculin A treatment has effects on traveling waves similar to hyper-osmolarity Fig.

However, this proposal does not exclude additional effects of actin. For instance, actin polymerization may push the membrane toward the substrate, increasing the membrane-substrate adhesion. We showed that our key conclusion was robust to this model variation Supplementary Fig. Alternatively, actin retrograde flow could remove F-BAR from the membrane.

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  • Introduction.

Dissecting more detailed molecular events of actin machinery in our traveling waves will be part of our future work. In sum, our work identifies membrane shape change as an important factor in determining the dynamics of traveling waves propagating along the cell cortex. This finding warrants closer scrutiny of the role of curvature sensing in other rhythmic cortical phenomena 2 , 3 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , possibly including actin-based cell polarity establishment 58 , 59 , 60 , 61 and cytokinesis For transient transfections, electroporation with Neon transfection system Life Technologies was used.

After transfection, cells were plated at subconfluent densities in mm glass bottom dishes MatTek, Ashland, MA or on round coverslips in a well plate overnight. In all experiments, cells were sensitized with mouse monoclonal anti-2,4-dinitrophenyl IgE Sigma-Aldrich at 0. DNA sequences corresponding to a. Constructs for the following proteins were kind gifts: Lifeact-mRuby from Dr. All plasmids were sequenced to vindicate their integrity. MetaMorph 7. Reflected light rays from two interfaces between coverslip and solution and between solution and plasma membrane interfered with each other, creating a bright or dark patch when they were constructive or destructive, respectively.

Prior to imaging, the coverslip was transferred to a custom perfusion chamber Chamlide, LCI placed on the heated stage of a Nikon Ti-E inverted microscope. A multi-valve perfusion control system MPS-8, LCI was used to switch rapidly between solutions flowing into the chamber and over the cells. The perfusion system was connected to a computer and controlled by MetaMorph software Molecular Device in synchronization with image acquisition.

An ImageJ-based software Fiji 63 was used to generate movies, kymographs, montages, and Z-stack projections. We determined the instantaneous wave speed and the cortical protein gradient at wavefront by the following procedures as illustrated in Supplementary Figs. To avoid complications arising from the interactions between multiple waves, we only chose well-defined single waves to determine the wave speed and the cortical protein gradient at the wavefront.

We set the boundaries of individual waves by determining where the image intensity fell to below a threshold slightly above the background intensity level Supplementary Fig. Next, the wave centroid r c t i of the segmented region for each time frame t i was calculated Supplementary Fig.