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Force requirements of endocytic vesicle formation

Marc Abella, Lynnel Andruck, Gabriele Malengo, Michal Skruzny

Preprint posted on November 11, 2020 https://www.biorxiv.org/content/10.1101/2020.11.11.378273v1

Measuring forces involved in endocytic vesicle formation.

Selected by Mariana De Niz

Categories: biophysics

Background

Mechanical forces are key for many cellular processes, including clathrin-mediated endocytosis. This is the major trafficking pathway transporting nutrients, signals, pathogens and plasma membrane components into the cell. During endocytosis, forces provided by endocytic proteins, formation of multiprotein scaffolds, protein crowding, and polymerization of the actin cytoskeleton reshape the plasma membrane into a vesicle. To transmit forces stored in the actin polymerization and crosslinking, actin filaments must be mechanically linked to the plasma membrane. This is accomplished by the force-transmitting protein linker made of endocytic adaptors Sla2-Ent1 in yeast, and Hip1R-epsin 1-3 in humans. These proteins bind cooperatively to the plasma membrane by N-terminal domains, and redundantly to actin filaments through C-terminal domains. The absence of Sla2/Hip1R-epsin linker severely impairs endocytosis. Assessing force requirements of endocytic membrane remodeling is essential for understanding endocytosis. In their work, Abella and colleagues (1) aimed to measure forces applied on the Sla2-Ent1 linker during endocytosis in yeast, using calibrated FRET-based tension sensor modules (TSMs) integrated into Sla2 protein. Overall, this study provides key force values and force profiles for understanding the mechanics of endocytosis and potentially other cellular membrane-remodeling processes.

 

Figure 1. Schematic of force requirements of endocytic vesicle formation measured by Sla2 force sensors.

Key findings and developments

The authors began by constructing yeast strains expressing various Sla2 force sensors-Sla2 fusion proteins containing different tension sensor modules (TSMs, ref. 2) connected by mTurquoise2 and mNeonGreen fluorophores. The TSMs sensitive to distinct force ranges: F40 (1-6 pN), HP35 (6-8 pN) or HP35st (9-11 pN) were inserted between the coiled-coil dimerization motif and the actin-binding THATCH domain of Sla2. To distinguish force-dependent from force-independent FRET changes, the authors also constructed strains with all TSMs integrated after the THATCH domain, at the C-terminus of Sla2.

To follow the forces applied on the Sla2 force sensors, the authors analysed FRET changes during individual endocytic events- by simultaneously recording the mTurquoise2 and mNeonGreen fluorescence signals (i.e. FRET ratio) at individual endocytic sites. A decreased ratio between mNeonGreen and mTurquoise2 fluorescence intensity during endocytic events, indicated force applied on all 3 Sla2 force sensors. FRET ratio profiles of all three Sla2 force sensors showed an initial decrease in the FRET ratio approximately 13 seconds before vesicle scission. This coincided with the appearance of fluorescence signal of the actin marker Abp1-mScarlet-I at the endocytic sites, indicating that the force applied over the Sla2 sensors correlates with the onset of actin polymerization at the endocytic site. The similar starting point of the FRET ratio drop and its subsequent stepwise decrease observed for all 3 sensors suggests that Sla2 molecules are sequentially recruited to the growing actin cytoskeleton during the course of endocytosis. Comparing the 3 force sensors showed that while the Sla2-F40 and Sla2-HP35 sensors behaved similarly, a smaller decrease in FRET ratio was observed for the Sla2-HP35st sensor (suited to detect higher forces). This indicates that the force applied over Sla2 therefore lies inside the dynamic range of HP35st (around 10 pN). This value, connected with the number of Sla2 molecules at the endocytic site (45-133, refs. 3 and 4) results in 450-1300 pN transmitted over the Sla2 linkers during a single endocytic event.

The authors then analysed the contributions of key membrane-remodeling factors to endocytic force transmission using the Sla2-HP35 sensor. They began by following the FRET ratio during endocytic events in cells deleted for protein Rvs167 (BAR-domain protein known to bind membrane invaginations during endocytosis). In these cells, membrane invagination is sometimes aborted and the membrane retracts back to its initial flat conformation without vesicle scission. The authors conclude that similar forces are applied on the Sla2 linker in WT cells and in Rvs167 mutants during initial membrane bending and early invagination, but following this, Rvs167 is crucial to facilitate productive transmission during the later invagination steps. Next, the authors analysed the role of the organization of the actin cytoskeleton by following the Sla2-HP35 sensor in cells lacking Bbc1- a negative regulator of actin polymerization at the endocytic site. Analysis of changes in the FRET ratio suggests that less force is transmitted over Sla2 in the last phase of invagination. This led the authors to conclude that there is extra force stored in the actin cytoskeleton of bbc1-mutant cells, which directly remodels the invaginating membrane.

The authors went on to investigate the roles of turgor pressure and plasma membrane tension during endocytosis. The internal turgor pressure of yeast cells is the main mechanical barrier counteracting endocytic membrane invagination. To compensate for this the cells were placed in hypertonic medium to lower the osmotic difference between the cell cytoplasm and the surrounding environment. This resulted in a smaller drop in the Sla2-HP35 FRET ratio, suggesting that less force is transmitted over the sensor under conditions reducing cellular turgor. This was also observed upon incubating the cells with a soluble lipid palmitoylcarnitine, which is incorporated into the yeast plasma membrane and reduces its tension. Together, these results suggest that a decrease of cell turgor pressure or plasma membrane tension reduce the force required for endocytosis.

Finally, the authors analysed the capacity of the endocytic force-transmitting machinery, by following Sla2 force sensors in cells incubated under hypotonic conditions, which should intensify cell turgor opposing endocytosis. For this, they used mutant cells that lack aquaglyceroporin and thus cannot adapt to hypoosmotic conditions by glycerol efflux. These cells containing Sla2-HP35 or Sla2-HP35st sensors were exposed to osmotic shifts. As osmolarity was lowered, the number of stalled endocytic events increased. However, despite these differences in numbers of stalled events, no significant difference was detected in the force transmitted over the Sla2-HP35st sensor during completed endocytic events. This indicates that there is no significant adaptation of the force-generating and/or transmitting system to hypoosmotic conditions. Altogether, the authors hypothesize that under hypotonic conditions, yeasts maintain the vital endocytic process by modulation of internal turgor and not by direct regulation of endocytic force-generating or transmitting machinery.

 

What I like about this preprint

I found the topic very interesting and relevant for various fields. As the authors themselves point out, endocytosis is the major trafficking pathway for nutrients, signals, pathogens and plasma membrane components into the cell- which is relevant to a plethora of research fields. I found also the methods implemented by the authors very interesting and novel.

 

References

  1. Abella et al, (2020), Force requirements of endocytic vesicle formation, bioRxiv.
  2. Cost, A.L., Khalaji, S., and Grashoff, C. (2019). Genetically Encoded FRET-Based Tension Sensors. Curr. Protoc. Cell Biol. 83, 1–29.
  3. Picco, A., Mund, M., Ries, J., Nédélec, F., and Kaksonen, M. (2015). Visualizing the functional architecture of the endocytic machinery. Elife 2015, 1–29.
  4. Sun, Y., Schöneberg, J., Chen, X., Jiang, T., Kaplan, C., Xu, K., Pollard, T.D., and Drubin, D.G. (2019). Direct comparison of clathrin-mediated endocytosis in budding and fission yeast reveals conserved and evolvable features. Elife 8:e50749.

 

Posted on: 18th December 2020

doi: https://doi.org/10.1242/prelights.26542

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Author's response

Michal Skruzny shared

Open questions 

1.You mentioned in your work that the force transmitting protein linker in human cells is Hip 1R-epsin 1-3. Is it equally feasible to study forces generated in mammalian cells during endocytosis, using the FRET-based method you explore here?

We hope so! Though the endocytic process is clearly more complex in mammalian cells, studies from Drubin, De Camilli and Garcia-Alai labs strongly suggest that conserved Hip1-epsin 1-3 linker is the major connector between the plasma membrane and the endocytic actin network. Already existing epsins-knockout murine cells (Messa et al., 2014) could be very valuable for such studies.

2.Do you think the various conclusions you reach in your work here would be conserved in mammalian cells? Do you think those forces might vary depending on the mammalian cell studied?

We think that our main conclusions regarding roles of hyper-/hypotonic conditions and plasma membrane tension will be similarly valid in mammalian systems as already suggested by studies pointing to their key role in endocytosis. These studies also indicate that forces required for endocytosis depend on the cell type and cell surrounding. We speculate that higher force might be needed for endocytosis in tissues and organs, where cells are stretched or tightly connected to the extracellular matrix.

3.You also mention in your work the relevance of endocytosis for various physiological processes including transporting nutrients, signals, pathogens and plasma membrane components into the cell. Would forces required for endocytic vesicle formation vary depending on the composition of the material being transported?

In yeast probably not, as endocytosis there copes much more with the high turgor barrier, not with any “difficult” cargo. However, in mammalian cells we have clear examples of differential endocytic uptake depending on the size or shape of the cargo as nicely shown e.g. for various viral particles (Cureton et al., 2010).

4.What alternative applications do you think your method for studying forces generated during endocytic vesicle formation, could have for other cell biology processes?

The field of cellular mechanobiology is very active and many labs study force-dependent processes inside and between cells. We hope that our approach shows the way, how to study these processes in a high temporal resolution and truly molecular level, which are prerequisites for determination of real force values and profiles for a given process. We believe that this is essential for mechanistic understanding, as well as for a potential use of many cellular processes in biomedicine or bioengineering.

5.Knowledge of force requirements for various cellular processes will be useful in many contexts. You particularly mention in your work, optimized drug delivery and selective molecular uptake. Is your method compatible with high-throughput, in order to be implemented into a pipeline (including perhaps automated image analysis) to investigate drug delivery and molecular uptake?

We think so, especially in our budding yeast system, which was many times proved to be very powerful for various high-throughput screens. The establishment of automated cell manipulation, image acquisition and analysis is clearly essential not only for potential uptake studies but also for further analyses of other proteins, biophysical conditions and signaling pathways involved. We are just putting our hands and heads in that!

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