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1.
Proc Natl Acad Sci U S A ; 121(25): e2318838121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38870057

RESUMO

Hertwig's rule states that cells divide along their longest axis, usually driven by forces acting on the mitotic spindle. Here, we show that in contrast to this rule, microtubule-based pulling forces in early Caenorhabditis elegans embryos align the spindle with the short axis of the cell. We combine theory with experiments to reveal that in order to correct this misalignment, inward forces generated by the constricting cytokinetic ring rotate the entire cell until the spindle is aligned with the cell's long axis. Experiments with slightly compressed mouse zygotes indicate that this cytokinetic ring-driven mechanism of ensuring Hertwig's rule is general for cells capable of rotating inside a confining shell, a scenario that applies to early cell divisions of many systems.


Assuntos
Caenorhabditis elegans , Fuso Acromático , Animais , Caenorhabditis elegans/embriologia , Camundongos , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Citocinese/fisiologia , Rotação , Zigoto/metabolismo , Zigoto/citologia , Zigoto/crescimento & desenvolvimento , Embrião não Mamífero/citologia , Desenvolvimento Embrionário/fisiologia , Modelos Biológicos
2.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35046036

RESUMO

Membranes of vacuoles, the lysosomal organelles of Saccharomyces cerevisiae (budding yeast), undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and vacuole membranes phase separate into micrometer-scale domains of two liquid phases. Recent studies suggest that these domains promote yeast survival by organizing membrane proteins that play key roles in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether cells regulate phase transitions in response to new physical conditions and how this occurs. Here, we measure transition temperatures and find that after an increase of roughly 15 °C, vacuole membranes appear uniform, independent of growth temperature. Moreover, populations of cells grown at a single temperature regulate this transition to occur over a surprisingly narrow temperature range. Remarkably, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. We isolate vacuoles from yeast during the rapid stage of growth, when their membranes do not natively exhibit domains. Ergosterol is the major sterol in yeast. We find that domains appear when ergosterol is depleted, contradicting the prevalent assumption that increases in sterol concentration generally cause membrane phase separation in vivo, but in agreement with previous studies using artificial and cell-derived membranes.


Assuntos
Membrana Celular/metabolismo , Saccharomyces cerevisiae/fisiologia , Ergosterol/metabolismo , Microdomínios da Membrana/metabolismo , Temperatura , Vacúolos/metabolismo
3.
Proc Natl Acad Sci U S A ; 118(50)2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34887356

RESUMO

Membrane invagination and vesicle formation are key steps in endocytosis and cellular trafficking. Here, we show that endocytic coat proteins with prion-like domains (PLDs) form hemispherical puncta in the budding yeast, Saccharomyces cerevisiae These puncta have the hallmarks of biomolecular condensates and organize proteins at the membrane for actin-dependent endocytosis. They also enable membrane remodeling to drive actin-independent endocytosis. The puncta, which we refer to as endocytic condensates, form and dissolve reversibly in response to changes in temperature and solution conditions. We find that endocytic condensates are organized around dynamic protein-protein interaction networks, which involve interactions among PLDs with high glutamine contents. The endocytic coat protein Sla1 is at the hub of the protein-protein interaction network. Using active rheology, we inferred the material properties of endocytic condensates. These experiments show that endocytic condensates are akin to viscoelastic materials. We use these characterizations to estimate the interfacial tension between endocytic condensates and their surroundings. We then adapt the physics of contact mechanics, specifically modifications of Hertz theory, to develop a quantitative framework for describing how interfacial tensions among condensates, the membrane, and the cytosol can deform the plasma membrane to enable actin-independent endocytosis.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Endocitose/fisiologia , Príons/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Membrana Celular , Proteínas do Citoesqueleto/genética , Citosol/fisiologia , Regulação Fúngica da Expressão Gênica , Glutamina/química , Mecanotransdução Celular , Conformação Proteica , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Substâncias Viscoelásticas
4.
Biophys J ; 122(6): 1043-1057, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36635960

RESUMO

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Lipidômica , Vacúolos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Lipídeos
5.
Small ; 19(22): e2206267, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36866488

RESUMO

Hybrid vesicles consisting of phospholipids and block-copolymers are increasingly finding applications in science and technology. Herein, small angle X-ray scattering (SAXS) and cryo-electron tomography (cryo-ET) are used to obtain detailed structural information about hybrid vesicles with different ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and poly(1,2-butadiene-block-ethylene oxide) (PBd22 -PEO14 , Ms  = 1800 g mol-1 ). Using single particle analysis (SPA) the authors are able to further interpret the information gained from SAXS and cryo-ET experiments, showing that increasing PBd22 -PEO14 mole fraction increases the membrane thickness from 52 Å for a pure lipid system to 97 Å for pure PBd22 -PEO14 vesicles. Two vesicle populations with different membrane thicknesses in hybrid vesicle samples are found. As these lipids and polymers are reported to homogeneously mix, bistability is inferred between weak and strong interdigitation regimes of PBd22 -PEO14 within the hybrid membranes. It is hypothesized that membranes of intermediate structure are not energetically favorable. Therefore, each vesicle exists in one of these two membrane structures, which are assumed to have comparable free energies. The authors conclude that, by combining biophysical methods, accurate determination of the influence of composition on the structural properties of hybrid membranes is achieved, revealing that two distinct membranes structures can coexist in homogeneously mixed lipid-polymer hybrid vesicles.


Assuntos
Bicamadas Lipídicas , Polímeros , Polímeros/química , Bicamadas Lipídicas/química , Espalhamento a Baixo Ângulo , Raios X , Difração de Raios X , Microscopia Eletrônica
6.
Proc Natl Acad Sci U S A ; 117(33): 19713-19719, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759217

RESUMO

Images of micrometer-scale domains in lipid bilayers have provided the gold standard of model-free evidence to understand the domains' shapes, sizes, and distributions. Corresponding techniques to directly and quantitatively assess smaller (nanoscale and submicron) liquid domains have been limited. Researchers commonly seek to correlate activities of membrane proteins with attributes of the domains in which they reside; doing so hinges on identification and characterization of membrane domains. Although some features of membrane domains can be probed by indirect methods, these methods are often constrained by the limitation that data must be analyzed in the context of models that require multiple assumptions or parameters. Here, we address this challenge by developing and testing two methods of identifying submicron domains in biomimetic membranes. Both methods leverage cryo-electron tomograms of ternary membranes under vitrified, hydrated conditions. The first method is optimized for probe-free applications: Domains are directly distinguished from the surrounding membrane by their thickness. This technique quantitatively and accurately measures area fractions of domains, in excellent agreement with known phase diagrams. The second method is optimized for applications in which a single label is deployed for imaging membranes by both high-resolution cryo-electron tomography and diffraction-limited optical microscopy. For this method, we test a panel of probes, find that a trimeric mCherry label performs best, and specify criteria for developing future high-performance, dual-use probes. These developments have led to direct and quantitative imaging of submicron membrane domains in vitrified, hydrated vesicles.


Assuntos
Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Bicamadas Lipídicas/química , Membrana Celular/química , Microdomínios da Membrana/química , Microdomínios da Membrana/ultraestrutura
7.
Langmuir ; 38(3): 1304-1310, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35026114

RESUMO

The first cell membranes were likely composed of single-chain amphiphiles such as fatty acids. An open question is whether fatty acid membranes could have functioned within evaporative lakes on the early Earth, which have been hypothesized to concentrate prebiotic reactants. Evaporation also concentrates monovalent salts, which in turn cause fatty acid membrane vesicles to flocculate; significant loss of encapsulated contents during flocculation would have impeded early cell evolution. Here, we tested whether fatty acid vesicles retain encapsulated contents after flocculation and after drying. We found that vesicles composed of 2:1 decanoic acid:decanol encapsulate calcein dye throughout a process of flocculation in saturated salt solution and subsequent disaggregation of vesicles by dilution of the salt. However, 30 minutes of complete dehydration disrupted encapsulation by fatty acid vesicles. In contrast, phospholipid vesicles maintained encapsulation. Our results reveal a selective pressure for protocells to incorporate phospholipids: while fatty acid membranes can retain encapsulated contents during periods of dilute and saturating salt, phospholipids are necessary for encapsulation during dry periods. Our results are consistent with the hypothesis that evaporative lakes were productive sites for prebiotic chemistry and the origin of cells.


Assuntos
Células Artificiais , Fosfolipídeos , Desidratação , Ácidos Graxos , Floculação , Humanos
8.
Proc Natl Acad Sci U S A ; 116(35): 17239-17244, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31405964

RESUMO

The membranes of the first protocells on the early Earth were likely self-assembled from fatty acids. A major challenge in understanding how protocells could have arisen and withstood changes in their environment is that fatty acid membranes are unstable in solutions containing high concentrations of salt (such as would have been prevalent in early oceans) or divalent cations (which would have been required for RNA catalysis). To test whether the inclusion of amino acids addresses this problem, we coupled direct techniques of cryoelectron microscopy and fluorescence microscopy with techniques of NMR spectroscopy, centrifuge filtration assays, and turbidity measurements. We find that a set of unmodified, prebiotic amino acids binds to prebiotic fatty acid membranes and that a subset stabilizes membranes in the presence of salt and Mg2+ Furthermore, we find that final concentrations of the amino acids need not be high to cause these effects; membrane stabilization persists after dilution as would have occurred during the rehydration of dried or partially dried pools. In addition to providing a means to stabilize protocell membranes, our results address the challenge of explaining how proteins could have become colocalized with membranes. Amino acids are the building blocks of proteins, and our results are consistent with a positive feedback loop in which amino acids bound to self-assembled fatty acid membranes, resulting in membrane stabilization and leading to more binding in turn. High local concentrations of molecular building blocks at the surface of fatty acid membranes may have aided the eventual formation of proteins.


Assuntos
Aminoácidos/química , Ácidos Graxos/química , Membranas Artificiais , Microscopia Crioeletrônica
9.
Chembiochem ; 21(19): 2764-2767, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358921

RESUMO

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.


Assuntos
Ácidos Decanoicos/química , Evolução Química , Nucleosídeos/química , RNA/química , Ribose/química , Sítios de Ligação , Ressonância Magnética Nuclear Biomolecular , Origem da Vida , Tamanho da Partícula
10.
Biophys J ; 115(4): 690-701, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30049406

RESUMO

Micron-scale, coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases are straightforward to observe in giant unilamellar vesicles (GUVs) composed of ternary lipid mixtures. Experimentally, uniform membranes undergo demixing when temperature is decreased: domains subsequently nucleate, diffuse, collide, and coalesce until only one domain of each phase remains. The sizes of these two domains are limited only by the size of the system. Under different conditions, vesicles exhibit smaller-scale domains of fixed sizes, leading to the question of what sets the length scale. In membranes with excess area, small domains are expected when coarsening is hindered or when a microemulsion or modulated phase arises. Here, we test predictions of how the size, morphology, and fluorescence levels of small domains vary with the membrane's temperature, tension, and composition. Using GUVs and cell-derived giant plasma membrane vesicles, we find that 1) the characteristic size of domains decreases when temperature is increased or membrane tension is decreased, 2) stripes are favored over circular domains for lipid compositions with low energy per unit interface, 3) fluorescence levels are consistent with domain registration across both monolayer leaflets of the bilayer, and 4) small domains form in GUVs composed of lipids both with and without ester-linked lipids. Our experimental results are consistent with several elements of current theories for microemulsions and modulated phases and inconsistent with others, suggesting a motivation to modify or enhance current theories.


Assuntos
Membrana Celular/química , Lipossomas Unilamelares/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animais , Cães , Células Madin Darby de Rim Canino , Pressão Osmótica , Temperatura
11.
Biophys J ; 113(6): 1200-1211, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28801104

RESUMO

A persistent challenge in membrane biophysics has been to quantitatively predict how membrane physical properties change upon addition of new amphiphiles (e.g., lipids, alcohols, peptides, or proteins) in order to assess whether the changes are large enough to plausibly result in biological ramifications. Because of their roles as general anesthetics, n-alcohols are perhaps the best-studied amphiphiles of this class. When n-alcohols are added to model and cell membranes, changes in membrane parameters tend to be modest. One striking exception is found in the large decrease in liquid-liquid miscibility transition temperatures (Tmix) observed when short-chain n-alcohols are incorporated into giant plasma membrane vesicles (GPMVs). Coexisting liquid-ordered and liquid-disordered phases are observed at temperatures below Tmix in GPMVs as well as in giant unilamellar vesicles (GUVs) composed of ternary mixtures of a lipid with a low melting temperature, a lipid with a high melting temperature, and cholesterol. Here, we find that when GUVs of canonical ternary mixtures are formed in aqueous solutions of short-chain n-alcohols (n ≤ 10), Tmix increases relative to GUVs in water. This shift is in the opposite direction from that reported for cell-derived GPMVs. The increase in Tmix is robust across GUVs of several types of lipids, ratios of lipids, types of short-chain n-alcohols, and concentrations of n-alcohols. However, as chain lengths of n-alcohols increase, nonmonotonic shifts in Tmix are observed. Alcohols with chain lengths of 10-14 carbons decrease Tmix in ternary GUVs of dioleoyl-PC/dipalmitoyl-PC/cholesterol, whereas 16 carbons increase Tmix again. Gray et al. observed a similar influence of the length of n-alcohols on the direction of the shift in Tmix. These results are consistent with a scenario in which the relative partitioning of n-alcohols between liquid-ordered and liquid-disordered phases evolves as the chain length of the n-alcohol increases.


Assuntos
Álcoois/química , Membrana Celular/química , Temperatura de Transição , Lipossomas Unilamelares/química , Álcoois/farmacologia , Animais , Fenômenos Biomecânicos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Microscopia , Fosfatidilcolinas/química , Ratos , Soluções , Água/química
12.
Biophys J ; 113(11): 2425-2432, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29211996

RESUMO

Controversy has long surrounded the question of whether spontaneous lateral demixing of membranes into coexisting liquid phases can organize proteins and lipids on micron scales within unperturbed, living cells. A clear answer hinges on observation of hallmarks of a reversible phase transition. Here, by directly imaging micron-scale membrane domains of yeast vacuoles both in vivo and cell free, we demonstrate that the domains arise through a phase separation mechanism. The domains are large, have smooth boundaries, and can merge quickly, consistent with fluid phases. Moreover, the domains disappear above a distinct miscibility transition temperature (Tmix) and reappear below Tmix, over multiple heating and cooling cycles. Hence, large-scale membrane organization in living cells under physiologically relevant conditions can be controlled by tuning a single thermodynamic parameter.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Sobrevivência Celular , Vacúolos/metabolismo , Leveduras/citologia
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