We thank Patricia Bassereau and Daniel L&#233;vy for their useful advice and discussions. This work benefited from the support of the ANR (Agence Nationale de la Recherche) for funding the project &#34;SEPTIME&#34;, ANR-13-JSV8-0002-01, ANR SEPTIMORF ANR-17-CE13-0014, and the project &#34;SEPTSCORT&#34;, ANR-20-CE11-0014-01. B. Chauvin is funded by the Ecole Doctorale &#34;ED564: Physique en Ile de France&#34; and Fondation pour lea Recherche M&#233;dicale. K. Nakazawa was supported by Sorbonne Universit&#233; (AAP Emergence). G.H. Koenderink was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO/OCW) through the &#8216;BaSyC-Building a Synthetic Cell&#39;. Gravitation grant (024.003.019).We thank the Labex Cell(n)Scale (ANR-11-LABX0038) and Paris Sciences et Lettres (ANR-10-IDEX-0001-02). We thank the Cell and Tissue Imaging (PICT-IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).

<ol>
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The authors have no conflicts of interest.

As stated above, a lipid mixture has been used that enhances PI(4,5)P2 incorporation within the lipid bilayer and thus facilitates septin-membrane interactions. Indeed, we have shown elsewhere25 that budding yeast septins interact with vesicles in a PI(4,5)P2-specific fashion. This lipid composition was adjusted empirically from screening multiple compositions and is now widely used by the authors. PI(4,5)P2 lipids have to be handled carefully. Stock solutions must...

Septins are cytoskeletal filament-forming proteins that interact with lipid membranes. Septins are ubiquitous in eukaryotes and essential to numerous cellular functions. They have been identified as the main regulators of cell division in budding yeast and mammals1,2. They are involved in membrane reshaping events, ciliogenesis3, and spermiogenesis4. Within mammalian cells, septins can also interact with actin and microtubules5,6,7 in a binder of Rho GTPases (BORG)-dependent manner8. In various tissues (neurons9, cilia3, spermatozoa10), septins have been identified as regulators of diffusion barriers for membrane-bound components11. Septins have also been shown to regulate membrane blebbing and protrusion formation12. Septins, being multi-tasking proteins, are implicated in the emergence of various prevalent diseases13. Their misregulation is associated with the emergence of cancers14 and neurodegenerative diseases15.
Depending on the organism, several septin subunits (two in Caenorhabditis elegans to 13 in humans) assemble to form complexes whose organization varies in a tissue-dependent fashion16. The basic septin building block gathers two to four subunits, present in two copies and self-assembled in a rod-like palindromic manner. In budding yeast, septins are octameric17,18. In situ, septins are often localized at sites with micrometer curvature; they are found at division constriction sites, at the base of cilia and dendrites, and at the annulus of spermatozoa19,20. At the membrane, the role of septins seems to be dual: they are implicated in reshaping the lipid bilayer and in maintaining membrane integrity21. Hence, investigating the biophysical properties of septin filament-forming proteins and/or subunits at the membrane is crucial for understanding their role. To dissect specific properties of septins in a well-controlled environment, bottom-up in vitro approaches are appropriate. So far, only a few groups have described the biophysical properties of septins in vitro20,22,23. Hence, as compared with other cytoskeletal filaments, the current knowledge on the behavior of septins in vitro remains limited.
This protocol describes how the organization of septin filaments, membrane reshaping, and curvature sensitivity can be analyzed19. To this end, a combination of optical and electron microscopy methods (fluorescence microscopy, cryo-electron microscopy [cryo-EM], and scanning electron microscopy [SEM]) has been used. The membrane reshaping of micrometer-sized giant unilamellar vesicles (GUVs) is visualized using fluorescence optical microscopy. The analysis of the arrangement and ultrastructure of septin filaments bound to lipid vesicles is performed using cryo-EM. Analysis of septin curvature sensitivity is carried out using SEM, by studying the behavior of septin filaments bound to solid-supported lipid bilayers deposited on wavy substrates of variable curvatures, which enables the analysis of curvature sensitivity for both positive and negative curvatures. As compared with previous analysis20,24, here, we propose to use a combination of methods to thoroughly analyze how septins can self-assemble, synergistically deform membrane, and be curvature-sensitive. This protocol is believed to be useful and adaptable to any filamentous protein that displays an affinity for membranes.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine</td><td>Avanti Polar Lipids</td><td>850725</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phospho-L-serine</td><td>Avanti Polar Lipids</td><td>840035</td><td></td></tr><tr><td>Bath sonicator</td><td>Elma</td><td></td><td>Elmasonic S10H</td></tr><tr><td>Bodipy-TR-Ceramide</td><td>invitrogen, Thermo Fischer scientific</td><td>11504726</td><td></td></tr><tr><td>Chemicals: NaCl, Tris-HCl, sucrose, KCl, MgCl2, B-casein, chloroform, sodium cacodylate, tannic acid, ethanol</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>nikon</td><td></td><td>spinning disk or confocal</td></tr><tr><td>Critical point dryer</td><td>Leica microsystems</td><td></td><td>CPD300</td></tr><tr><td>Deionized water generator</td><td>MilliQ</td><td>F1CA38083B</td><td>MilliQ integral 3</td></tr><tr><td>Egg L-&amp;alpha;-phosphatidylcholine</td><td>Avanti Polar Lipids</td><td>840051</td><td></td></tr><tr><td>Field Emission Gun SEM (FESEM)</td><td>Carl Zeiss</td><td></td><td>Gemini SEM500</td></tr><tr><td>Glutaraldehyde 25 %, aqueous solution</td><td>Thermo Fischer scientific</td><td>50-262-19</td><td></td></tr><tr><td>High vacuum grease, Dow corning</td><td>VWR</td><td></td><td></td></tr><tr><td>IMOD software</td><td>https://bio3d.colorado.edu/imod/</td><td></td><td>software suite for tilted series image alignment and 3D reconstruction</td></tr><tr><td>Lacey Formvar/carbon electron microscopy grids</td><td>Eloise</td><td>01883-F</td><td></td></tr><tr><td>Lipids</td><td>Avanti Polar Lipids</td><td></td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate</td><td>Avanti Polar Lipids</td><td>840046</td><td></td></tr><tr><td>Metal evaporator</td><td>Leica microsystems</td><td></td><td>EM ACE600</td></tr><tr><td>NOA (Norland Optical Adhesives), NOA 71 and NOA 81</td><td>Norland Products</td><td>NOA71, NOA81</td><td></td></tr><tr><td>Osmium tetraoxyde 4%</td><td>delta microscopies</td><td>19170</td><td></td></tr><tr><td>Osmometer</td><td>L&amp;ouml;ser</td><td></td><td>15 M</td></tr><tr><td>Plasma cleaner</td><td>Alcatel</td><td></td><td>pascal 2005 SD</td></tr><tr><td>Plasma generator</td><td>Electron Microscopy Science</td><td></td><td></td></tr><tr><td>Plunge freezing equipment</td><td>leica microsystems</td><td></td><td>EMGP</td></tr><tr><td>Transmission electron microscope</td><td>Thermofischer</td><td></td><td>Tecnai G2 200 kV, LaB6</td></tr><tr><td>Uranyl acetate</td><td>Electron Microscopy Science</td><td>22451</td><td>this product is not available for purchase any longer</td></tr><tr><td>Wax plates, Vitrex</td><td>VWR</td><td></td><td></td></tr></tbody></table>

bottom-up in vitro methods to assay the ultrastructural organization, membrane reshaping, and curvature sensitivity behavior of septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

GUVs deformations 
Typical confocal fluorescence images of GUVs reshaped after being incubated with septins are displayed in Figure 3, in conditions where septins polymerize. Bare GUVs (Figure 3A) were perfectly spherical. Upon incubation with more than 50 nM budding yeast septin filaments, the vesicles appeared deformed. Up to a concentration of 100 nM budding yeast septin octamers, the vesicles appeared facetted, and the deformations rema...

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Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Septins are cytoskeletal proteins. They interact with lipid membranes and can sense but also generate membrane curvature at the micron scale. We describe in this protocol bottom-up in vitro methodologies for analyzing membrane deformations, curvature-sensitive septin binding, and septin filament ultrastructure.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

bottom-up-vitro-methods-to-assay-ultrastructural-organization

Research

JoVE Journal

Biology

2.4K Views.  Sorbonne Université. Met behulp van dit protocol visualiseerden we de organisatie van cytoskeletale filamenteuze eiwitten op patroonsubstraten met verschillende krommingen. We kunnen de krommingsgevoeligheid testen. De resolutie van scanning elektronenmicroscoopbeelden is hoog genoeg zodat nanometerschaalorganisaties worden gevisualiseerd.De methoden die we gebruiken voor fixatie zijn mild genoeg om de intra-structurele organisatie van het eiwit te behouden. Dit blijft in het kader van fundamenteel onderzo...

Video: Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric ...

Biochemistry

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays&#160;and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

Medicine

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics

Source: Curtis, B. N. et al. <a href="https://www.jove.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022)
In this video, we demonstrate a procedure to investigate septin organization on a glass-supported lipid bilayer in vitro using total internal reflection fluorescence microscopy.

Source: Curtis, B. N. et al. Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022)
In this ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Source: Curtis, B. N. et al., <a href="https://www.jove.com/v/64090">Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions.</a> J. Vis. Exp. (2022).
This video describes the technique of studying the septin protein assembly on microspheres coated with lipid bilayers. This technique helps in understanding the curvature-dependent assemblies of protein on curved surfaces.

Source: Curtis, B. N. et al., Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions. J. Vis. Exp. (2022).
This ...

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

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