A liquid phase of synapsin and lipid vesicles

Milovanović, Dragomir; Wu, Yumei; Bian, Xin; Camilli, Pietro De

doi:10.1126/science.aat5671

Cited by 424 publications

(486 citation statements)

References 38 publications

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“…Previous studies suggested that the IDLs of adjacent SSB molecules can interact with each other to enhance the cooperativity of ssDNA binding, while the IDL was also proposed to interact with the OB fold Kozlov et al, 2015Kozlov et al, , 2017. Moreover, the central role played by similar low-sequence-complexity, Gln/Gly/Pro-rich IDRs in previously investigated LLPS systems (Bakthavachalu et al, 2018;Mannen et al, 2016;Milovanovic et al, 2018;Sabari et al, 2018) as well as the predicted LLPS propensities suggest a role for the IDL in SSB LLPS ( Fig. 1B-D).…”

Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 58%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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Bacterial single stranded (ss) DNA-binding proteins (SSB) are essential for the replication and maintenance of the genome. SSBs share a conserved ssDNA-binding domain, a less conserved intrinsically disordered linker (IDL) and a highly conserved C-terminal peptide (CTP) motif that mediates a wide array of protein-protein interactions with DNA-metabolizing proteins. Here we show that the E. coli SSB protein forms liquid-liquid phase separated condensates in cellular-like conditions through multifaceted interactions involving all structural regions of the protein. SSB, ssDNA and SSBinteracting molecules are highly concentrated within the condensates, whereas phase separation is overall regulated by the stoichiometry of SSB and ssDNA. Together with recent results on subcellular SSB localization patterns, our results point to a conserved mechanism by which bacterial cells store a pool of SSB and SSB-interacting proteins. Dynamic phase separation enables rapid mobilization of this protein pool to protect exposed ssDNA and repair genomic loci affected by DNA damage. RESULTS SSB forms dynamic LLPS condensates in physiologically relevant conditions in vitroDue to the fact that SSB shares several features with hitherto described LLPS drivers, we sought to determine if LLPS could be a yet undiscovered capability of SSB facilitating its multifaceted roles in genome maintenance. We performed a bioinformatics analysis that revealed that E. coli SSB, in particular its IDL region, shows high propensity for LLPS, according to multiple dedicated sequencebased prediction methods (Bolognesi et al., 2016;Lancaster et al., 2014;Vernon et al., 2018) (Fig. 1B-D). This feature of SSB is universally conserved among bacteria, as the majority of analyzed SSBs (72.1 %) harbored by representative bacterial species from 15 large phylogenetic groups show similar LLPS propensities (Tables S1-2; Fig. 1E).In line with its predicted LLPS propensity, purified E. coli SSB (30 µM; SSB concentrations are expressed as those of tetramer molecules throughout the paper) forms an opaque, turbid solution at low NaCl concentration (50 mM) in vitro at room temperature and also at the native temperature of E. coli cells (37°C) ( Fig. 2A-B). The process is reversible as elevation of NaCl concentration to 200 mM leads to immediate loss of turbidity. The Clconcentration in E. coli cells depends on their environment (Schultz et al., 1962); however, while the exact value is not known, it can be assumed to be in a similar range (5-100 mM) as that measured for the cells of mammalian E. coli hosts, whereas the major metabolic anion in bacterial cells is glutamate (Glu; 100 mM) (Bennett et al., 2009). Strikingly, SSB forms a turbid solution even in 200 mM NaGlu ( Fig. 2A-B). (It must be noted that experiments using NaGlu always contained a fixed amount of 20 mM NaCl, for technical reasons.) Investigation of the protein solution by differential interference contrast (DIC) microscopy after diluting SSB into low-salt buffer revealed the formation of regular spherical drop...

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Section: Multivalent Interactions Between Ssb Idl Regions Are Requirementioning

confidence: 58%

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Harami

Kovács

Pancsa

et al. 2019

Preprint

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“…During prolonged stimulation, when the SV recycling process becomes rate limiting, SVs are mobilized from these clusters to ensure reliable SV availability. It was recently proposed that SVs form clusters by phase separation; specifically, the SV peripheral phosphoprotein synapsin‐1 was shown to be able to organize SVs in clusters by liquid–liquid phase separation . Synapsin is an abundant protein and can reversibly associate with SVs .…”

Section: Coordination Between Vesicle Acidification and Trafficking Amentioning

confidence: 99%

“…It was recently proposed that SVs form clusters by phase separation; specifically, the SV peripheral phosphoprotein synapsin-1 was shown to be able to organize SVs in clusters by liquid-liquid phase separation. 74 Synapsin is an abundant protein and can reversibly F I G U R E 3 Hypothetical model of vATPase recycling within the SV recycling pathway. Endocytic retrieval from the plasma membrane by clathrin-dependent mechanism results in the formation of clathrin-coated vesicles, which contain a fully assembled vATPase complex.…”

Section: Coordination Between Vesicle Acidification and Traffickingmentioning

confidence: 99%

Regulation of synaptic vesicle acidification at the neuronal synapse

Gowrisankaran

Milošević

2020

IUBMB Life

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The vacuolar H+‐adenosine triphosphatases (vATPases) acidify multiple intracellular organelles, including synaptic vesicles (SVs) and secretory granules. Acidification of SVs represents a critical point during the SV cycle: without acidification, neurotransmitters cannot be loaded into SVs. Despite the obvious importance of the vesicle acidification process for neurotrasmission and the life of complex organisms, little is known about the regulation of vATPase at the neuronal synapse. In addition, the composition of the vATPase complex on the SVs is unclear. Here, we summarize the key features of vATPase found on SVs, and propose a model of how vATPase activity is regulated during the SV cycle. It is anticipated that the information from the SV lumen is communicated to SV surface in order to signal successful acidification and neurotransmitter loading: we postulate here that the regulators of the vATPase activity exist (e.g., Rabconnectin‐3) that promote the recruitment of SV peripheral proteins and, consequently, SV fusion.

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“…Moreover, condensates control where reactions occur by clustering molecules or membrane‐bound organelles in specific locations. The clustering of synaptic vesicles and neurotransmitter receptors at presynaptic and postsynaptic regions is mediated by the phase separation of proteins such as Synapsin in the presynapse and SynGAP and PSD‐95 in the postsynapse . Synaptonemal complex condensates control the spatial patterning of meiotic recombination factors …”

Section: Biomolecular Condensates: Assembly Function and Regulationmentioning

confidence: 99%

Biomolecular condensates in neurodegeneration and cancer

Spannl

Tereshchenko

Mastromarco

et al. 2019

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The intracellular environment is partitioned into functionally distinct compartments containing specific sets of molecules and reactions. Biomolecular condensates, also referred to as membrane‐less organelles, are diverse and abundant cellular compartments that lack membranous enclosures. Molecules assemble into condensates by phase separation; multivalent weak interactions drive molecules to separate from their surroundings and concentrate in discrete locations. Biomolecular condensates exist in all eukaryotes and in some prokaryotes, and participate in various essential house‐keeping, stress‐response and cell type‐specific processes. An increasing number of recent studies link abnormal condensate formation, composition and material properties to a number of disease states. In this review, we discuss current knowledge and models describing the regulation of condensates and how they become dysregulated in neurodegeneration and cancer. Further research on the regulation of biomolecular phase separation will help us to better understand their role in cell physiology and disease.

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A liquid phase of synapsin and lipid vesicles

Cited by 424 publications

References 38 publications

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Phase separation by ssDNA binding protein controlledviaprotein-protein and protein-DNA interactions

Regulation of synaptic vesicle acidification at the neuronal synapse

Biomolecular condensates in neurodegeneration and cancer

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