07:52:49 From Andrej Kosmrlj to Everyone: https://online.kitp.ucsb.edu/online/films21/ 08:08:21 From Reinhard Lipowsky to Everyone: Question to Patricia: 08:08:45 From Reinhard Lipowsky to Everyone: Question to Patricia: 08:11:01 From Reinhard Lipowsky to Everyone: Qu to Patricia B: what about the orientation of the pumps and channels in the GUVs? Did these membrane proteins have a preferred orientation or was the orientation more or less random? 08:11:42 From Patricia Bassereau to Everyone: The way we prepare the GUVs most of the time, it is roughly 50-50 08:13:14 From Markus Deserno to Everyone: Those clusters look a little bit like the beginnings of a “diffusion limited aggregation” cluster. That might be testable, since those are basically stuck once they form. But I think yours are a lot more dynamic, which would rule out plain DLA. 08:17:14 From Rumiana Dimova to Everyone: To ask a question live, please raise hand in Zoom (accessed from "Reactions") 08:24:25 From Cecilia Leal to Everyone: The group of Charles Schroeder has developed what is called a stoke trap that controls GUVs with flow, can pull tubes without touching them. 08:25:27 From Cecilia Leal to Everyone: https://www.pnas.org/content/113/15/3976 08:26:28 From Alfredo Sciortino to Everyone: but one is still interfering by using flow right.? 08:27:11 From Rumiana Dimova to Everyone: Yes, this is still quite an external perturbation 08:27:24 From Hammad Faizi to Everyone: @ Cecilia with Stokes trap can we measure changes in effective tension? 08:27:26 From Markus Deserno to Everyone: I don’t think interfering with the system is intrinsically bad, as long as you know what that interference is doing. 08:27:36 From Petia Vlahovska to Everyone: Agreed with Alfredo. The flow induces tension! 08:28:00 From Alfredo Sciortino to Everyone: (and I also agree with Markus of course..!) 08:29:01 From Cecilia Leal to Everyone: Right, there interference but it is less than a pipette. @hammad, you measure ether fluctuation spectra like normal but the vesicles are trapped in flow 08:30:09 From Hammad Faizi to Everyone: @Cecilia the changes in tension imposed may over power the changes by for example pumps activity? And we may not detect it? I don't know. 08:32:53 From Cecilia Leal to Everyone: @hammad, we are doing those experiments right now. You can measure the fluctuations of regular GUVs, if activity increases the fluctuations, I expect that we will be able to measure them 08:33:13 From Rumiana Dimova to Everyone: If you pull a tube and measure its diameter with super-resolution microscopy, you will be able to measure the membrane tension provided you know what the membrane bending rigidity is. We have demonstrated this here: https://pubs.acs.org/doi/10.1021/acs.nanolett.9b05232 Although we did use pipettes, we demonstrate that measuring only the tube diameter is sufficient. 08:33:20 From Hammad Faizi to Everyone: @Cecilia. That is exciting! Keep us updated. 08:36:48 From Petia Vlahovska to Everyone: @Cecilia: The effect of the flow on the fluctuations could be tricky… There is a paper by Carlos that might be relevant https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.92.108103 08:37:13 From Ali Azadbakht to Everyone: @Rumy, isn't so tricky to pull a tube from a floppy vesicle in the order of less than 10^-7 N/m? I believe even these tiny tubes could change the tension in order of magnitude. 08:37:49 From Cecilia Leal to Everyone: Thanks @Petia. Anyway, I think this community might find the stokes trap an interesting tool to look at to study GUV elastic properties. In the PNAS paper above the technique is introduced and in this paper it is applied to GUVs: https://pubs.rsc.org/en/content/articlelanding/2020/sm/c9sm02048a#!divAbstract 08:40:56 From Padmini Rangamani to Everyone: Questions for Patricia: Thank you for the great talk. Following up on what Reinhard talked about. I was also wondering, what do you think could be happening to the thickness of the bilayers due to conformation changes of the proteins (channels/pumps/motors)? Is it possible that the changes at the length scale of the bilayer thickness could be dominant? From a theory perspective, I'm thinking of distension-dominant effects as a possibility. Or even lipid tilt. Any thoughts? 08:42:05 From Padmini Rangamani to Everyone: I think this paper could be relevant to some of the ideas we are discussing: not GUVs but supported lipid bilayers: https://www.pnas.org/content/108/17/6975.short 08:45:35 From Martin Michael Müller to Everyone: Question to Patricia/Markus: Does the volume of the GUV change? 08:46:09 From Buddhapriya Chakrabarti to Everyone: What he is saying is that you turn on the activity 08:46:14 From Buddhapriya Chakrabarti to Everyone: After it is in equilibrium 08:46:50 From Buddhapriya Chakrabarti to Everyone: That would require correlated behaviour of proteins though @Markus 08:50:56 From Pietro Cicuta to Everyone: I think Cecile was right on the aspiration experiment. The tension is just clamped because it is DeltaP set by the hydrostatic mismatch, combined with the curvature of the capillary, which does not change. So when the vesicle has more activity, it pulls out more membrane from the capillary, and fluctuates more, which is consistent with higher T_eff. All at constant tension. 08:52:39 From Markus Deserno to Everyone: But aren’t these fluctuations a source of entropic tension? 08:53:22 From Pietro Cicuta to Everyone: Yes the entropy is one of the contributions to the overall tension. 08:54:07 From Rumiana Dimova to Everyone: Changing thickness can be done with photo-active lipids for example 08:54:08 From Marino Arroyo to Everyone: In line with @Markus question and a fixed tension ensemble, can membrane proteins enter the membrane tongue inside of the pipette? If not, the contact line between pipette and free vesicle is some kind of semi-permeable interface that allows passage of lipids and not proteins. This would mean a different notion of tension inside and outside of the pipette, since one would include the osmotic tension of proteins and the other not. Does anyone know if this effect matters in interpreting the experiments? 08:55:59 From Pietro Cicuta to Everyone: @Marino - nice one to check experimentally! I would imagine proteins can diffuse. But there are papers that show the clustering of proteins where the membrane is flatter, and/or where there are fewer fluctuations.... so maybe there is a partitioning in/out of the capillary. 08:59:10 From Markus Deserno to Everyone: Seems we have ample material for an extended coffee session, or some other specialized event, on tension in active systems. 😀 09:02:01 From Rikhia Ghosh to Everyone: Can one think of reduction of tension (-ve sign) in active membrane in terms of membrane getting compressed or lipids coming closer to each other compared to a passive membrane? 09:02:15 From Martin Michael Müller to Everyone: Another idea is that the expansion of the proteins leads to excess area: Bastien Loubet, Udo Seifert, and Michael Andersen Lomholt Phys. Rev. E 85, 031913 2012 09:03:36 From Rumiana Dimova to Everyone: We should try to move this discussion on tension to Slack 09:11:19 From Pietro Cicuta to Everyone: Flicker itself needs to be done carefully - in this paper with Matthew Turner we showed that considering the optical projection brings the numbers closer to the scattering. S.A. Rautu, D. Orsi, L. Di Michele, G. Rowlands, P. Cicuta, M.S. Turner The Role of Optical Projection in the Analysis of Membrane Fluctuations Soft Matter 13, 3480-3483 (2017) but I don't know why pipette could be different! 09:15:30 From Rumiana Dimova to Everyone: @Pietro: Yes, one needs to do fluctuation analysis very carefully, we give some very detailed tips here also regarding Matthew Turner's paper https://pubs.rsc.org/en/content/articlelanding/2020/SM/D0SM00943A#!divAbstract 09:18:25 From Hammad Faizi to Everyone: https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.124.158102 09:18:33 From Hammad Faizi to Everyone: Alfredo is talking about this paper 09:19:30 From Alfredo Sciortino to Everyone: yes, thanks Hammad. And also https://www.nature.com/articles/s41586-020-2730-x#Sec18 09:22:25 From TRIPTA BHATIA to Everyone: Patricia: there are two different slopes in Helene paper with Ipsen and Mouritsen, at lower and higher modes. There is a slide on this in your talk. Related to the discussion about different slopes.. Regards 09:22:27 From Alex Klotz to Everyone: no experiment is very quick :'( 09:26:54 From Alex Klotz to Everyone: Side question for people who are familiar with membrane channels: are there any mechanisms by which cells pass *circular* DNA like plasmids through membranes, besides intracell pili? 09:28:07 From Edward Lyman to Everyone: But is 40-50% area coverage of proteins the integral membrane part? Or does it include soluble domains proximal to the membrane? I usually find answers more like 20-30% when asking about cross sectional/integral membrane area. After all, most integral membrane proteins are single pass TM helices. 09:29:47 From Maria-Anna Kirmpaki to Everyone: @alex kotz nuclear pores in the eykaryotic cells at least are used in order to import or export genetic material. Importers for entry exporters for exit 09:31:54 From Alex Klotz to Everyone: do they send circular molecules as well as linear? 09:32:42 From Patricia Bassereau to Everyone: @Ed: You are probably right. I have these numbers in mind, just to estimate crowding for in plane diffusion. 09:33:22 From Maria-Anna Kirmpaki to Everyone: @alex Klotz no but endosomes through endocytosis could deliver plasmids. through fusion with the membrane 09:34:34 From Sarah Keller to Everyone: Amplifying Ed’s comment, Allison Dupuy and Don Engelman (www.pnas.org􏰱cgi􏰱doi􏰱10.1073􏰱pnas.0712379105) found 23% of membrane area of red blood cells was proteins. Of course, domains outside of the membrane can also interact to keep proteins from coming closer. As only one example, the research of Jeanne Stachowiak is very nice. 09:35:50 From Richard Pastor to Everyone: john - we lst you 09:43:05 From Maria-Anna Kirmpaki to Everyone: @alex Klotz https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705778/ . Please check this paper, so eventually seems that apart from linear DNA circular plasmids can as well enter the nucleus through the pores. 09:51:32 From Patricia Bassereau to Everyone: @Tripta: Rony discussed diffusion in active membranes as far I remember, not time correlation fonction of fluctuations. Helène Bouvrais measured 2 times in time correlation 09:52:14 From Buddhapriya Chakrabarti to Everyone: Good luck with elastic theories in getting that :) 09:52:41 From TRIPTA BHATIA to Everyone: @Patricia: thanks 10:02:51 From Anand Srivastava to Everyone: Thanks all. 10:02:52 From Sam Hess to Everyone: Thank you very much 10:02:53 From Vikram Jadhao to Everyone: Thank you.