The synthetic pathways of life’s building blocks are envisaged to be through a series of complex prebiotic reactions and processes. However, the strategy to compartmentalize and concentrate biopolymers under prebiotic conditions remains elusive. Liquid-liquid phase separation is a mechanism by which membraneless organelles form inside cells, and has been hypothesized as a potential mechanism for prebiotic compartmentalization. Associative phase separation of oppositely charged species has been shown to partition RNA, but the strongly negative charge exhibited by RNA suggests that RNA-polycation interactions could inhibit RNA folding and its functioning inside the coacervates. Here, we present a prebiotically plausible pathway for non-associative phase separation within an evaporating all-aqueous sessile droplet. We quantitatively investigate the kinetic pathway of phase separation triggered by the non-uniform evaporation rate, together with the Marangoni flow-driven hydrodynamics inside the sessile droplet. With the ability to undergo liquid-liquid phase separation, the drying droplets provide a robust mechanism for formation of prebiotic membraneless compartments, as demonstrated by localization and storage of nucleic acids, in vitro transcription, as well as a three-fold enhancement of ribozyme activity. The compartmentalization mechanism illustrated in this model system is feasible on wet organophilic silica-rich surfaces during early molecular evolution.

During the podosome formation, distinct phosphatidylinositol 3,4,5-trisphosphate lipid (PI(3,4,5)P3) production and F-actin polymerization take place at integrin-mediated adhesions. Membrane-associated actin regulation factors, such as myosin-1, serve as key molecules to link phosphatidylinositol signals to podosome assembly. Here, we report that long-tailed myosin-1e (Myo1e) is enriched at the ventral layer of the podosome core in a PI(3,4,5)P3 dependent manner. Combination of TH1 and TH2 (TH12) of Myo1e tail domains contains the essential motif for PI(3,4,5)P3-dependent membrane association and ventral localization at the podosome. TH12 KR2A (K772A and R782A) becomes dissociated from the plasma membrane. While F-actin polymerizations are initialized from the ventral layer of the podosome, TH12 precedes the recruitment of N-WASP and Arp2/3 in the initial phase of podosome formation. Over-expression of TH12, not TH12 KR2A impedes PI(3,4,5)P3 signaling, restrains F-actin polymerization, and inhibits podosome formation. TH12 also suppresses gelatin degradation and migration speed of invadopodia-forming A375 melanoma cells. Thus, TH12 domain of Myo1e serves as a regulatory component to connect phosphatidylinositol signaling to F-actin polymerization at the podosome. Movie S1: Photo-conversion of cytosolic mEOS2-β-actin reveals the assembly process of F-actin at the ventral layer of the podosome in MEF-Src. Spot activation of 405nm laser is applied at the circled area away from podosome arc, and photo-converted cytosolic mEOS2-β-actin can freely diffuse with the cell. After photo-conversion, 561nm excitation is used to monitor the incorporation of photo-converted actin into different layers of the podosome by a spinning disk confocal microscope. At first, newly converted mEOS2-actin incorporate into the ventral layer (z= 0μm) and then gradually move to the upper layers of podosome. Scale bar represents 10μm. Download Original Video (.6 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s03.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482de742a767b9626fc43b45aea11d177d0759a81799f9c1e6b6ab957fcd8d573a66b1d7315355fd719be91c4d3eac47faa382095688d5a Movie S2: PI(3,4,5)P3 lipids are enriched at the podosome. PH-BTK and PH-PLCδ are used as specific probes to visualize the distribution of PI(3,4,5)P3 and PI(4,5)P2 lipids, respectively. During the podosome formation in MEF-Src, PH-BTK is enriched at the F-actin core in the podosome, whereas the level of PH-PLCδ is reduced. Scale bar represents 10μm. Download Original Video (7.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s04.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482d9742a767b9626fc074dbc37fef13a2af8fc286a0633bbd27f99082a59c69a81bed09faaaa9bbfac2beb65bf92f9b7923a1967a30698 Movie S3: Fluorescence recovery after photobleaching (FRAP) of \ full-length Myo1e and various tail constructs at the podosome in MEF-Src. The recovery of full-length Myo1e is slow. Different from full-length Myo1e, TH1, TH2, and TH3 all have fast recoveries after photobleaching. TH12 and TH123, on the other hand, exhibit similar slow recoveries as full-length Myo1e. See Fig. S5C for the statistical information. Scale bar represents 10μm. Download Original Video (8.1 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s05.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482d8742a767b9626fc7898e46057150ab3728191c8f2a03a97cd01f0e09113c8f0d79de9107306b1b5279b1562e5b4e1d87832ec516ab8 Movie S4: The expression of TH12 suppresses the formation of podosome rosettes and arcs. F-actin polymerization at the podosome becomes short-lived and unstable in TH12-expressed MEF-Src. Scale bar represents 10μm. Download Original Video (4.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s06.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482db742a767b9626fc9b3a0b7832780f064975fd008b40524ca62b680bb748c940f1953d1fc09034b09dcbf998ba98d1482fc69fe7e750 Movie S5: Dynamical assembly of podosomes in RAW 264.7 macrophage. Myo1e is localized at the F-actin core in each podosome. Scale bar represents 10μm. Download Original Video (3.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s07.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482da742a767b9626fc463f7110801f04c1c4f78fd5ecb2b57187f321533876c2be2e51b79b27969c89ee0414d1937d9bd33e833af535cc Movie S6: The expression of TH12 disrupts the podosome formation in RAW 264.7 macrophage. F-actin fails to assemble distinct podosomes. Scale bar represents 10μm. Download Original Video (7.7 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-06-0398/20181230/media/mc-e18-06-0398-s08.,1200,900,600,300,150,.mp4.m3u8?b92b4ad1b4f274c70877518410abb28bf45a6fadfff54c0884ea58110075615c1d5919ea753725730a4097154a8f56c8491b2e70b0e87b5e9f35b4d61387d2130c8df350edbabb355e8ef985f87fe75bc1a3492f8b9b2cd072aa362005914caa2bb55e55385abc7c311a3d64c314be416f8226297e525482d5742a767b9626fc8c96569d6cfffb7297b96a5a22069b2eecfbfa2197be3170cee2cdd9e7ec31db330c2772100cf46ed2b2b1721f04