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Membrane Proteins: Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.

Reconstitution of Membrane Proteins

JoVE 5693

Reconstitution is the process of returning an isolated biomolecule to its original form or function. This is particularly useful for studying membrane proteins, which enable important cellular functions and affect the behavior of nearby lipids. To study the function of purified membrane proteins in situ, they must be reconstituted by integrating them into an artificial lipid membrane.

 Biochemistry

Protein Associations

JoVE 10704

The cell membrane—or plasma membrane—is an ever-changing landscape. It is described as a fluid mosaic as various macromolecules are embedded in the phospholipid bilayer. Among the macromolecules are proteins. The protein content varies across cell types. For example, mitochondrial inner membranes contain ~76%, while myelin contains ~18% protein content. Individual cells contain many types ofbrane proteins—red blood cells contain over 50—and different cell types harbor distinct membrane protein sets. Membrane proteins have wide-ranging functions. For example, they can be channels or carriers that transport substances, enzymes with metabolic roles, or receptors that bind to chemical messengers. Like membrane lipids, most membrane proteins contain hydrophilic (water-loving) and hydrophobic (water-fearing) regions. The hydrophilic areas are exposed to water-containing solution inside the cell, outside the cell, or both. The hydrophobic regions face the hydrophobic tails of phospholipids within the membrane bilayer. Membrane proteins can be classified by whether they are embedded (integral) or associated with the cell membrane (peripheral). Most integral proteins are transmembrane proteins, which traverse both phospholipid layers, spanning the entire membrane. Their hydrophilic regions extend from both sides of the membrane, facing cytosol on

 Core: Membranes and Cellular Transport

What are Membranes?

JoVE 10971

A key characteristic of life is the ability to separate the external environment from the internal space. To do this, cells have evolved semi-permeable membranes that regulate the passage of biological molecules. Additionally, the cell membrane defines a cell’s shape and interactions with the external environment. Eukaryotic cell membranes also serve to compartmentalize the internal space into organelles, including the endomembrane structures of the nucleus, endoplasmic reticulum and Golgi apparatus. Membranes are primarily composed of phospholipids composed of hydrophilic heads and two hydrophobic tails. These phospholipids self-assemble into bilayers, with tails oriented toward the center of the membrane and heads positioned outward. This arrangement allows polar molecules to interact with the heads of the phospholipids both inside and outside of the membrane but prevents them from moving through the hydrophobic core of the membrane. Proteins and carbohydrates contribute to the unique properties of a cell’s membrane. Integral proteins are embedded in the membrane, while peripheral proteins are attached to either the internal or external surface of the membrane. Transmembrane proteins are integral proteins that span the entire cell membrane. Transmembrane receptor proteins are important for communicating messages from the outside to the insid

 Core: Membranes and Cellular Transport

Facilitated Transport

JoVE 10705

The chemical and physical properties of plasma membranes cause them to be selectively permeable. Since plasma membranes have both hydrophobic and hydrophilic regions, substances need to be able to transverse both regions. The hydrophobic area of membranes repel substances such as charged ions. Therefore, such substances need special membrane proteins to cross a membrane successfully. In the process of facilitated transport, also known as facilitated diffusion, molecules and ions travel across a membrane via two types of membrane transport proteins: channels and carrier proteins. These membrane transport proteins enable diffusion without requiring additional energy. Channel proteins form a hydrophilic pore through which charged molecules can pass through, thus avoiding the hydrophobic layer of the membrane. Channel proteins are specific for a given substance. For example, aquaporins are channel proteins that specifically facilitate the transport of water through the plasma membrane. Channel proteins are either always open or gated by some mechanism to control flow. Gated channels remain closed until a particular ion or substance binds to the channel, or some other mechanism occurs. Gated channels are found in the membranes of cells such as muscle cells and nerve cells. Muscle contractions occur when the relative concentrations of ions on the interior and

 Core: Membranes and Cellular Transport

Cell-surface Biotinylation Assay

JoVE 5647

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or “recycled” back to the membrane. The cell surface biotinylation assay provides researchers with a way to study…

 Cell Biology

The Extracellular Matrix

JoVE 10695

In order to maintain tissue organization, many animal cells are surrounded by structural molecules that make up the extracellular matrix (ECM). Together, the molecules in the ECM maintain the structural integrity of tissue as well as the remarkable specific properties of certain tissues.

The extracellular matrix (ECM) is commonly composed of ground substance, a gel-like fluid, fibrous components, and many structurally and functionally diverse molecules. These molecules include polysaccharides called glycosaminoglycans (GAGs). GAGs occupy most of the extracellular space and often take up a large volume relative to their mass. This results in a matrix that can withstand tremendous forces of compression. Most GAGs are linked to proteins—creating proteoglycans. These molecules retain sodium ions based on their positive charge and therefore attract water, which keeps the ECM hydrated. The ECM also contains rigid fibers such as collagens—the primary protein component of the ECM. Collagens are the most abundant proteins in animals, making up 25% of protein by mass. A large diversity of collagens with structural similarities provide tensile strength to many tissues. Notably, tissue like skin, blood vessels, and lungs need to be both strong and stretchy to perform their physiological role. A protein called elastin gives p

 Core: Cell Structure and Function

Protein Crystallization

JoVE 5689

Protein crystallization, obtaining a solid lattice of biomolecules, elucidates protein structure and enables the study of protein function. Crystallization involves drying purified protein under a combination of many factors, including pH, temperature, ionic strength, and protein concentration. Once crystals are obtained, the protein structure can be elucidated by x-ray diffraction and…

 Biochemistry

Viral Structure

JoVE 10822

Viruses are extraordinarily diverse in shape and size, but they all have several structural features in common. All viruses have a core that contains a DNA- or RNA-based genome. The core is surrounded by a protective coat of proteins called the capsid. The capsid is composed of subunits called capsomeres. The capsid and genome-containing core are together known as the nucleocapsid.

Many criteria are used to classify viruses, including capsid design. Most viruses have icosahedral or helical capsids, although some viruses have developed more complex capsid structures. The icosahedral shape is a 20-sided, quasi-spherical structure. Rhinovirus, the virus that causes the common cold, is icosahedral. Helical (i.e., filamentous or rod-shaped) capsids are thin and linear, resembling cylinders. The nucleic acid genome fits inside the grooves of the helical capsid. Tobacco mosaic virus, a plant pathogen, is a classic example of a helical virus. Some viruses have capsids that are enclosed by an envelope of lipids and proteins outside of the capsid. This viral envelope is not produced by the virus but is acquired from the host’s cell. These envelope molecules protect the virus and mediate interactions with the host’s cells. The viral capsid not only protects the virus’s genome, but it also plays a critical role in interactions with host cells. For i

 Core: Viruses

An Introduction to Endocytosis and Exocytosis

JoVE 5646

Cells can take in substances from the extracellular environment by endocytosis and actively release molecules into it by exocytosis. Such processes involve lipid membrane-bound sacs called vesicles. Knowledge of the molecular architecture and mechanisms of both is key to understanding normal cell physiology, as well as the disease states that arise when they become…

 Cell Biology

Annexin V and Propidium Iodide Labeling

JoVE 5650

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death—either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a…

 Cell Biology

What is an Electrochemical Gradient?

JoVE 10699

Adenosine triphosphate, or ATP, is considered the primary energy source in cells. However, energy can also be stored in the electrochemical gradient of an ion across the plasma membrane, which is determined by two factors: its chemical and electrical gradients.

The chemical gradient relies on differences in the abundance of a substance on the outside versus the inside of a cell and flows from areas of high to low ion concentration. In contrast, the electrical gradient revolves around an ion’s electrical charge and the overall charges of the intracellular and extracellular environments. The electrical gradient of a positively-charged ion flows from positive to negative regions, while the reverse is true for negatively-charged ions. It is the combined action of these electrical and chemical factors that determine the ultimate direction of an electrochemical gradient. When an ion moves along this path, down its electrochemical gradient, energy is freed that can then power diverse biological processes.

 Core: Membranes and Cellular Transport

Secondary Active Transport

JoVE 10707

One example of how cells use the energy contained in electrochemical gradients is demonstrated by glucose transport into cells. The ion vital to this process is sodium (Na+), which is typically present in higher concentrations extracellularly than in the cytosol. Such a concentration difference is due, in part, to the action of an enzyme “pump” embedded in the cellular membrane that actively expels Na+ from a cell. Importantly, as this pump contributes to the high concentration of positively-charged Na+ outside a cell, it also helps to make this environment “more positive” than the intracellular region. As a result, both the chemical and electrical gradients of Na+ point towards the inside of a cell, and the electrochemical gradient is similarly directed inwards. Sodium-glucose cotransporters (SGLTs) exploit the energy stored in this electrochemical gradient. These proteins, primarily located in the membranes of intestinal or kidney cells, help in the absorption of glucose from the lumen of these organs into the bloodstream. In order to function, both an extracellular glucose molecule and two Na+ must bind to the SGLT. As Na+ migrates into a cell through the transporter, it travels with its electrochemical gradient, expelling energy that the protein uses to move glucose ins

 Core: Membranes and Cellular Transport

An Introduction to Cellular and Molecular Neuroscience

JoVE 5213

Cellular and molecular neuroscience is one of the newest and fastest growing subdisciplines in neuroscience. By investigating the influences of genes, signaling molecules, and cellular morphology, researchers in this field uncover crucial insights into normal brain development and function, as well as the root causes of many pathological conditions.


 Neuroscience

Neuronal Transfection Methods

JoVE 5215

Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in cells. Because this method can be used to silence the expression of specific proteins or to drive the expression of foreign or modified proteins, transfection is an extremely useful tool in the study of the cellular and…

 Neuroscience

Gap Junctions

JoVE 10986

Multicellular organisms employ a variety of ways for cells to communicate with each other. Gap junctions are specialized proteins that form pores between neighboring cells in animals, connecting the cytoplasm between the two, and allowing for the exchange of molecules and ions. They are found in a wide range of invertebrate and vertebrate species, mediate numerous functions including cell differentiation and development, and are associated with numerous human diseases, including cardiac and skin disorders. Vertebrate gap junctions are composed of transmembrane proteins called connexins (CX), and six connexins form a hemichannel called a connexon. Humans have at least 21 different forms of connexins that are expressed in almost all cell types. A connexon hemichannel is said to be homomeric when all six connexins are the same, and heteromeric when composed of different types. Most cells express more than one type of connexin. These can form functional connexon hemichannels or a full gap junction channel by pairing up with a counterpart on an adjacent cell. The gap junctions are considered homotypic when each connexon is the same, and heterotypic when they differ. Clusters called gap junction plaques often form where the channels are continually recycled and degraded at the center of the plaques and replaced at the periphery. Gap junctions allow the p

 Core: Cell Structure and Function

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

1Department of Biological Sciences, Markey Center for Structural Biology, Purdue University, 2National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, 3National Institute of General Medical Sciences (NIGMS), National Institutes of Health

JoVE 53245

 Chemistry

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

1Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 2Department of Genetics and Complex Diseases, T. H. Chan School of Public Health, Harvard Medical School, 3Department of Cell Biology, Harvard Medical School, 4Sorbonne Universités, UPMC University Paris 06, 5Center for Studies in Physics and Biology, The Rockefeller University

JoVE 56086

 Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

1Institut National de la Santé et de la Recherche Médicale, UMR 631, Parc scientifique de Luminy, 2Centre National de la Recherche Scientifique, UMR 6102, Parc scientifique de Luminy, 3Centre d'Immunologie de Marseille-Luminy, Aix-Marseille University, 4École Centrale Marseille, Technopôle de Château-Gombert, 5Institut Fresnel, Aix-Marseille University, 6Centre National de la Recherche Scientifique, UMR 6133, Aix-Marseille University

JoVE 3599

 Biology

Image Processing Protocol for the Analysis of the Diffusion and Cluster Size of Membrane Receptors by Fluorescence Microscopy

1Department of Macromolecular Structures, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 2Campus Urb. Montepríncipe s/n, Univ. San Pablo CEU, 3Department of Immunology and Oncology, Centro Nacional de Biotecnología, Campus Univ. Autónoma de Madrid, 4Department of Cell Signaling, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CSIC), 5Meyer Cancer Center, 6Department of Anatomy and Cell Biology, McGill Univ.

JoVE 59314

 Immunology and Infection

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health

JoVE 52281

 Biology

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS)

JoVE 54349

 Neuroscience

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

1Centre for Molecular Medicine Norway, Faculty of Medicine, University of Oslo, 2Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Oslo, 3Department of Chemistry and Chemical Engineering, Chalmers University of Technology

JoVE 58923

 Bioengineering
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