Cell Membrane Manipulation By Scramblases

Since the seminal paper describing the cell membranes via the fluid-mosaic model [1], there has been continued interest in membrane composition and its physiological importance.  Soon after, it was first hypothesized that the lipids within the membranes existed in an asymmetric state, which was shown using intact vs compromised red blood cells[2].  Importantly, it was shown that phospholipid composition, in particular, phosphatidylserine (PS) and phosphatidyl ethanolamine (PE) localize primarily on the cytoplasmic leaflet of the plasma membrane.  Conversely, other phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SP) are concentrated on the outer leaflet. Passive, transverse movement of phospholipids occurs on the scale of many hours, and it is unlikely the distinct asymmetric pattern of phospholipids could be achieved without a catalytic mediator (reviewed in [3]).  This led to the postulation that active transporters likely existed, and soon thereafter ATP-dependent lipid pumps like flippases and floppases were discovered. Additionally, ATP-independent but Ca2+-dependent lipid channels were also identified, and unlike the lipid pumps which move specific phospholipids in a specific direction [4], scramblases move phospholipids bidirectionally (see figure 1).  Lipid asymmetry is critical for many physiologic processes, so one may wonder what the benefit of diminishing asymmetry via scramblases is. Below we discuss the physiologic role of scramblases and their structural features.
Structural Analysis of TMEM Scramblases 
The TMEM16 family is comprised of Ca2+-activated chloride channels and scramblase channels that share a homodimeric architecture; within each protomer there are 10 transmembrane helices (reviewed in [5]). Brunner et al. showed that in nhTMEM16, helices 3-7 form a unique groove-like structure that is important for the transfer of lipids across membranes [6].  Interestingly, this groove is open to the membrane core but is hydrophilic alluding to the possibility as the pathway for lipid permeation.  Several studies have confirmed that this groove is important for lipid scrambling whereby lipid headgroups enter and diffuse through this hydrophilic groove while their tails remain within the hydrophobic core of the membrane (reviewed in [5]). More recently, it was shown that Ca2+ binding in nhTMEM16 induced a stepwise transition that transitions the catalytic groove from a closed state (shielded from the membrane) to an open state (accessible to lipid headgroups)[7]. Previous studies with mTMEM16F showed that it existed in a closed state even under maximal scramblase activity; however, a recent study suggested that massive molecular dynamic examination was needed to observe its true state.  In this study, they found that distal Ca2+ binding allosterically affected the groove region and promoted an open hydrophilic structure during scrambling activity [8].  Another recent study by Falzone et al. showed that an open hydrophilic groove is not a requirement for scrambling by the family member, afTMEM16; rather, this scramblase performs membrane thinning as a mechanism to enable phospholipid scrambling [9]. The authors suggest that this mechanism may be relevant to other scramblases that do not have an open groove structure.  Advancements in cryo-EM have enabled investigators to study these complex scramblase proteins to better understand how their discrete structural differences affect their functions.

Figure Legend: The three types of phospholipid translocases and their directional movement of phospholipids

Scramblases Regulate Critical Cellular Events
The kinetics by which flippases/floppases and scramblases function is vastly different.  Due to the active movement of phospholipids against an equilibrium gradient the necessary energy requirements and conformational changes results in a slower movement of phospholipids by flippases/floppases relative to scramblases (reviewed in [3]).  Scramblases allow for the rapid change in phospholipids across its channels that can quickly abolish asymmetry.  These types of drastic changes are necessary during critical cellular events.  For example, Suzuki et al. identified that the Xkr8 scramblase plays a critical role in apoptosis, a form of highly regulated programmed cell death [10].  When Xkr8 is activated through caspase cleavage it results in the rapid translocation of PS to the outer leaflet that functions as an “eat me” signal by macrophages.  This event is a hallmark of apoptosis and is important for programmed death as engulfment by macrophages minimizes the release of toxic cytoplasmic substances that would be harmful to neighboring cells.  Xkr8 was recently shown to be activated in a caspase-independent mechanism that utilizes phosphorylation and targets critical sites near the caspase cleavage site on the protein [11]. Another well-known role for scramblases is their effect on blood coagulation [12].  During secondary hemostasis, a scramblase known as transmembrane protein 16F (TMEM16F) promotes loss of lipid asymmetry through PS exposure on the outer leaflet.  Abundant PS exposure on the outer leaflet on platelets recruits clotting factors that lead to the activation of thrombin and the formation of an insoluble fibrin clot [13].  More recently, TMEM16F was shown to promote PS exposure during trophoblast fusion in a calcium-dependent fashion, and if the PS signal was suppressed, cell fusion was blocked [14].  Knockout of TMEM16F using CRISPR-Cas9 methods completely blocked trophoblast fusion, which was rescued with the reintroduction of TMEM16F.  Other TEMEM scramblases have been identified in several genetic diseases, neurological disorders, and even cancer (reviewed in [5]).
Summary and Future Directions
Asymmetric composition of phospholipids in the cell membrane is important for normal physiologic function, and acute changes by scramblases are observed in critical cellular events.  In another example, a recent study showed how the SARS-CoV-2 promotes an increase in Ca2+ levels, activation of TMEM16F (ANO6), exposure of PS, and enhanced fusion of the viral and cell membranes [15].  The group identified a compound A6-001 with high potency and selectivity to TMEM16F, and treatment with the compound blunted SARS-CoV-2 replication.  Furthermore, novel scramblases continue to be identified, such as, the protein CLPTM1L that functions to translocate glucosaminylphatidylinositols from the cytosol to the lumen of ER and is critical for biosynthesis of GPIs which function as membrane anchors [16].  Clearly, there is much to learn about this critical family of lipid regulating proteins, their impact on membrane composition, and how they affect critical cellular processes.  Cytoskeleton has an array of live cell membrane probes to aide in cell membrane investigation; such as, the novel Flipper-TR membrane tension probes, the MemGlow polarity probes, and the fluorogenic MemGlow membrane labeling probes. 



1. Singer, S.J. and G.L. Nicolson, The fluid mosaic model of the structure of cell membranes. Science, 1972. 175(4023): p. 720-31.
2. Bretscher, M.S., Asymmetrical lipid bilayer structure for biological membranes. Nat New Biol, 1972. 236(61): p. 11-2.
3. Clarke, R.J., K.R. Hossain, and K. Cao, Physiological roles of transverse lipid asymmetry of animal membranes. Biochim Biophys Acta Biomembr, 2020. 1862(10): p. 183382.
4. Bishop, W.R. and R.M. Bell, Assembly of the endoplasmic reticulum phospholipid bilayer: the phosphatidylcholine transporter. Cell, 1985. 42(1): p. 51-60.
5. Falzone, M.E., et al., Known structures and unknown mechanisms of TMEM16 scramblases and channels. J Gen Physiol, 2018. 150(7): p. 933-947.
6. Brunner, J.D., et al., X-ray structure of a calcium-activated TMEM16 lipid scramblase. Nature, 2014. 516(7530): p. 207-12.
7. Kalienkova, V., et al., Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM. Elife, 2019. 8.
8. Khelashvili, G., et al., The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase. Commun Biol, 2022. 5(1): p. 990.
9. Falzone, M.E., et al., TMEM16 scramblases thin the membrane to enable lipid scrambling. Nat Commun, 2022. 13(1): p. 2604.
10. Suzuki, J., et al., Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science, 2013. 341(6144): p. 403-6.
11. Sakuragi, T., H. Kosako, and S. Nagata, Phosphorylation-mediated activation of mouse Xkr8 scramblase for phosphatidylserine exposure. Proc Natl Acad Sci U S A, 2019. 116(8): p. 2907-2912.
12. Suzuki, J., et al., Calcium-dependent phospholipid scrambling by TMEM16F. Nature, 2010. 468(7325): p. 834-8.
13. Zwaal, R.F., P. Comfurius, and E.M. Bevers, Surface exposure of phosphatidylserine in pathological cells. Cell Mol Life Sci, 2005. 62(9): p. 971-88.
14. Zhang, Y., et al., TMEM16F phospholipid scramblase mediates trophoblast fusion and placental development. Sci Adv, 2020. 6(19): p. eaba0310.
15. Sim, J.R., et al., Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition. Cell Rep, 2022. 40(3): p. 111117.
16. Wang, Y., et al., Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis. Proc Natl Acad Sci U S A, 2022. 119(14): p. e2115083119.