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Cell Membrane: The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.

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

The Resting Membrane Potential

JoVE 10845

The relative difference in electrical charge, or voltage, between the inside and the outside of a cell membrane, is called the membrane potential. It is generated by differences in permeability of the membrane to various ions and the concentrations of these ions across the membrane.

The membrane potential of a cell can be measured by inserting a microelectrode into a cell and comparing the charge to a reference electrode in the extracellular fluid. The membrane potential of a neuron at rest—that is, a neuron not currently receiving or sending messages—is negative, typically around -70 millivolts (mV). This is called the resting membrane potential. The negative value indicates that the inside of the membrane is relatively more negative than the outside—it is polarized. The resting potential results from two major factors: selective permeability of the membrane, and differences in ion concentration inside the cell compared to outside. Cell membranes are selectively permeable because most ions and molecules cannot cross the lipid bilayer without help, often from ion channel proteins that span the membrane. This is because the charged ions cannot diffuse through the uncharged hydrophobic interior of membranes. The most common intra- and extracellular ions found in the nervous tissue are potassium (K+), sodium (Na+…

 Core: Nervous System

Cell Structure- Concept

JoVE 10587

Background

Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19th century, the Cell Theory was proposed to define a cell, which states:



Every living organism is made up of one or more cells.
The cells…

 Lab Bio

Cell-surface Signaling

JoVE 10877

Hormones—or any molecule that binds to a receptor, known as a ligand—that are lipid-insoluble (water-soluble) are not able to diffuse across the cell membrane. In order to be able to affect a cell without entering it, these hormones bind to receptors on the cell membrane. When a first messenger, a hormone, binds to a receptor, a signal cascade is set off, causing second messengers, proteins inside the cell, to become activated, resulting in downstream effects. Cell membrane receptors have three portions: an external ligand-binding domain, a transmembrane domain, and an internal domain. There are three categories of cell membrane receptors based on the consistency of the structure and function of these domains within each category. One category is ligand-gated ion channels which, when bound to a ligand, undergo a conformational change, allowing ions through a channel formed by the transmembrane portion of the receptor. A second category is G-proteins-coupled receptors which have a distinct structure with seven transmembrane domains. Binding of the external domain to a ligand causes the alpha subunit, one of three subunits attached to the internal portion of the receptor, to disassociate from the receptor and create a cellular response. The third category of receptors, the enzyme-linked receptor—also called catalytic receptor

 Core: Endocrine System

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

Magnetic Activated Cell Sorting (MACS): Isolation of Thymic T Lymphocytes

JoVE 10495

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France
2 INSERM U1223, Paris, France
3 Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, …

 Immunology

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

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

Receptor-mediated Endocytosis

JoVE 10708

Receptor-mediated endocytosis is a process through which bulk amounts of specific molecules can be imported into a cell after binding to cell surface receptors. The molecules bound to these receptors are taken into the cell through inward folding of the cell surface membrane, which is eventually pinched off into a vesicle within the cell. Structural proteins, such as clathrin, coat the budding vesicle and give it its round form. One well-characterized example of receptor-mediated endocytosis is the transport of low-density lipoproteins (LDL cholesterol) into the cell. LDL binds to transmembrane receptors on the cell membrane. Adapter proteins allow clathrin to attach to the inner surface of the membrane. These protein complexes bend the membrane inward, creating a clathrin-coated vesicle inside the cell. The neck of the endocytic vesicle is pinched off from the membrane by a complex of the protein dynamin and other accessory proteins. The endocytic vesicle fuses with an early endosome, and the LDL dissociates from the receptor proteins due to a lower pH environment. Empty receptor proteins are separated into transport vesicles to be re-inserted into the outer cell membrane. LDL remains in the endosome, which binds with a lysosome. The lysosome provides digestive enzymes that break up LDL into free cholesterol that can be used by the cell. There ar

 Core: Membranes and Cellular Transport

Ion Channels

JoVE 10722

Ion channels maintain the membrane potential of a cell. For most cells, especially excitable ones, the inside has a more negative charge than the outside of the cell, due to a greater number of negative ions than positive ions. For excitable cells, like firing neurons, contracting muscle cells, or sensory touch cells, the membrane potential must be able to change rapidly moving from a negative membrane potential to one that is more positive. To achieve this, cells rely on two types of ion channels: ligand-gated and voltage-gated. Ligand-gated ion channels, also called ionotropic receptors, are transmembrane proteins that form a channel but which also have a binding site. When a ligand binds to the surface, it opens the ion channel. Common ionotropic receptors include the NMDA, kainite, and AMPA glutamate receptors and the nicotinic acetylcholine receptors. When a ligand, like glutamate or acetylcholine, binds to its receptor it allows the influx of sodium (Na+) and calcium (Ca++) ions into the cells. The positive ions, or cations, follow down their electrochemical gradient, moving from the more positive extracellular surface to the less positive (more negative) intracellular surface. This changes the membrane potential near the receptor, which can then activate nearby voltage gated ion channels to propagate the change in membrane potential throughout the cell

 Core: Cell Signaling

Action Potentials

JoVE 10844

Neurons communicate by firing action potentials—the electrochemical signal that is propagated along the axon. The signal results in the release of neurotransmitters at axon terminals, thereby transmitting information in the nervous system. An action potential is a specific “all-or-none” change in membrane potential that results in a rapid spike in voltage.

Neurons typically have a resting membrane potential of about -70 millivolts (mV). When they receive signals—for instance, from neurotransmitters or sensory stimuli—their membrane potential can hyperpolarize (become more negative) or depolarize (become more positive), depending on the nature of the stimulus. If the membrane becomes depolarized to a specific threshold potential, voltage-gated sodium (Na+) channels open in response. Na+ has a higher concentration outside of the cell as compared to the inside, so it rushes in when the channels open, moving down its electrochemical gradient. As positive charge flows in, the membrane potential becomes even more depolarized, in turn opening more channels. As a result, the membrane potential quickly rises to a peak of around +40 mV. At the peak of the action potential, several factors drive the potential back down. The influx of Na+ slows because the Na+ channels start to inactiv

 Core: Nervous System

Diffusion and Osmosis- Concept

JoVE 10622

Cell Membranes and Diffusion

In order to function, cells are required to move materials in and out of their cytoplasm via their cell membranes. These membranes are semipermeable, meaning that certain molecules are allowed to pass through, but not others. This movement of molecules is mediated by the phospholipid bilayer and its embedded proteins, some of which act as transport channels…

 Lab Bio

Primary Active Transport

JoVE 10706

In contrast to passive transport, active transport involves a substance being moved through membranes in a direction against its concentration or electrochemical gradient. There are two types of active transport: primary active transport and secondary active transport. Primary active transport utilizes chemical energy from ATP to drive protein pumps that are embedded in the cell membrane. With energy from ATP, the pumps transport ions against their electrochemical gradients—a direction they would not normally travel by diffusion. To understand the dynamics of active transport, it is important to first understand electrical and concentration gradients. A concentration gradient is a difference in the concentration of a substance across a membrane or space that drives movement from areas of high concentration to areas of low concentration. Similarly, an electrical gradient is the force resulting from the difference between electrochemical potentials on each side of the membrane that leads to the movement of ions across the membrane until the charges are similar on both sides of the membrane. An electrochemical gradient is created when the forces of a chemical concentration gradient and electrical charge gradient are combined. One important transporter responsible for maintaining the electrochemical gradient in cells is the sodium-potassium pump. The pr

 Core: Membranes and Cellular Transport

Bacterial Signaling

JoVE 10713

At times, a group of bacteria behaves like a community. To achieve this, they engage in quorum sensing, the perception of higher cell density that results in a shift in gene expression. Quorum sensing involves both extracellular and intracellular signaling. The signaling cascade starts with a molecule called an autoinducer (AI). Individual bacteria produce AIs that move out of the bacterial cell membrane into the extracellular space. AIs can move passively along a concentration gradient out of the cell, or be actively transported across the bacterial membrane. When cell density in the bacterial populations is low, the AIs diffuse away from the bacteria, keeping the environmental concentration of AIs low. As bacteria reproduce and continue to excrete AIs, the concentration of AIs increases, eventually reaching a threshold concentration. This threshold permits AIs to bind membrane receptors on the bacteria, triggering changes in gene expression across the whole bacterial community. Many bacteria are broadly classified as gram positive or gram negative. These terms refer to the color that the bacteria take on when treated with a series of staining solutions which were developed by Hans Christian Joachim Gram over a century ago. If bacteria pick up a purple color, they are gram-positive; if they look red, they are gram-negative. These stain colors are pic

 Core: Cell Signaling

Hair Cells

JoVE 10854

Hair cells are the sensory receptors of the auditory system—they transduce mechanical sound waves into electrical energy that the nervous system can understand. Hair cells are located in the organ of Corti within the cochlea of the inner ear, between the basilar and tectorial membranes. The actual sensory receptors are called inner hair cells. The outer hair cells serve other functions, such as sound amplification in the cochlea, and are not discussed in detail here. Hair cells are named after the hair-like stereocilia that protrude from their tops and touch the tectorial membrane. The stereocilia are arranged by height and are attached by thin filaments called tip links. The tip links are connected to stretch-activated cation channels on the tips of the stereocilia. When a sound wave vibrates the basilar membrane, it creates a shearing force between the basilar and tectorial membranes that moves the hair cell stereocilia from side to side. When the cilia are displaced towards the tallest cilium, the tip links stretch, opening the cation channels. Potassium (K+) then flows into the cell, because there is a very high concentration of K+ in the fluid outside of the stereocilia. This large voltage difference creates an electrochemical gradient that causes an influx of K+ once the channels are opened. This influx o

 Core: Sensory Systems

Patch Clamp Electrophysiology

JoVE 5202

Neuron cell membranes are populated with ion channels that control the movement of charge into and out of the cell, thereby regulating neuron firing. One extremely useful technique for investigating the biophysical properties of these channels is called patch clamp recording. In this method, neuroscientists place a polished glass micropipette against a cell and apply…

 Neuroscience

Pinocytosis

JoVE 10709

Cells use energy-requiring bulk transport mechanisms to transfer large particles, or large amounts of small particles, into or out of the cell. The cells envelop the particles in spherical membranes called vesicles or vacuoles. Vesicles that transport material into the cell are built from the cell membrane. These vesicles encapsulate external molecules and transport them into the cell in a process called endocytosis. Pinocytosis (“cellular drinking”) is one of three main types of endocytosis. In pinocytosis, the cell repeatedly takes in fluid from the surrounding environment using tiny vesicles. Pinocytosis occurs in many cell types. In the small intestine, bristle-like protrusions called microvilli use pinocytosis to absorb nutrients from food. Egg cells use pinocytosis to obtain nutrients before fertilization. In pinocytosis and other forms of endocytosis, vesicles form when sections of the cell membrane sink inward, creating tear-shaped pockets that surround the material being taken into the cell. In pinocytosis, the imported material consists of fluid and other molecules. As the membrane reconnects, the vesicles pinch off, separating from the membrane. In the process, the vesicles enter the cell, taking the enclosed substances with them. Specific characteristics distinguish pinocytosis from the other forms of endocytosis&mdash

 Core: Membranes and Cellular Transport

What are Viruses?

JoVE 10821

A virus is a microscopic infectious particle that consists of an RNA or DNA genome enclosed in a protein shell. It is not able to reproduce on its own: it can only make more viruses by entering a cell and using its cellular machinery. When a virus infects a host cell, it removes its protein coat and directs the host’s machinery to transcribe and translate its genetic material. The hijacked cell assembles the replicated components into thousands of viral progeny, which can rupture and kill the host cell. The new viruses then go on to infect more host cells. Viruses can infect different types of cells: bacteria, plants, and animals. Viruses that target bacteria, called bacteriophages (or phages), are very abundant. Current research focuses on phage therapy to treat multidrug-resistant bacterial infections in humans. Viruses that infect cultivated plants are also highly studied since epidemics lead to huge crop and economic losses. Viruses were first discovered in the 19th century when an economically-important crop, the tobacco plant, was plagued by a mysterious disease—later identified as Tobacco mosaic virus. Animal viruses are of great importance both in veterinary research and in medical research. Moreover, viruses underlie many human diseases, ranging from the common cold, chickenpox, and herpes, to more dangerous infection

 Core: Viruses

Mitosis and Cytokinesis

JoVE 10762

In eukaryotic cells, the cell's cycle—the division cycle—is divided into distinct, coordinated cellular processes that include cell growth, DNA replication/chromosome duplication, chromosome distribution to daughter cells, and finally, cell division. The cell cycle is tightly regulated by its regulatory systems as well as extracellular signals that affect cell proliferation. The processes of the cell cycle occur over approximately 24 hours (in typical human cells) and in two major distinguishable stages. The first stage is DNA replication, during the S phase of interphase. The second stage is the mitotic (M) phase, which involves the separation of the duplicated chromosomes into two new nuclei (mitosis) and cytoplasmic division (cytokinesis). The two phases are separated by intervals (G1 and G2 gaps), during which the cell prepares for replication and division. Mitosis can be divided into five distinct stages—prophase, prometaphase, metaphase, anaphase, and telophase. Cytokinesis, which begins during anaphase or telophase (depending on the cell), is part of the M phase, but not part of mitosis. As the cell enters mitosis, its replicated chromosomes begin to condense and become visible as threadlike structures with the aid of proteins known as condensins. The mitotic spindle apparatus b

 Core: Cell Cycle and Division

Types of Hormones

JoVE 10988

Hormones can be classified into three main types based on their chemical structures: steroids, peptides, and amines. Their actions are mediated by the specific receptors they bind to on target cells.

Steroid hormones are derived from cholesterol and are lipophilic in nature. This allows them to readily traverse the lipid-rich cell membrane to bind to their intracellular receptors in the cytoplasm or nucleus. Once bound, the cytoplasmic hormone-receptor complex translocates to the nucleus. Here, it binds to regulatory sequences on the DNA to alter gene expression. Peptide hormones are made up of chains of amino acids and are hydrophilic. Hence, they are unable to diffuse across the cell membrane. Instead, they bind to extracellular receptors present on the surface of target cells. Such binding triggers a series of signaling reactions within the cell to ultimately carry out the specific functions of the hormone. Amine hormones are derived from a single amino acid, either tyrosine or tryptophan. This class of hormones is unique because they share their mechanism of action with both steroid as well as peptide hormones. For example, although epinephrine and thyroxine are both derived from the amino acid tyrosine, they mediate their effects through diverse mechanisms. Epinephrine binds to G-protein coupled receptors present on the surface of the plasma membran

 Core: Endocrine System

What are Second Messengers?

JoVE 10720

Because many receptor binding ligands are hydrophilic, they do not cross the cell membrane and thus their message must be relayed to a second messenger on the inside. There are several second messenger pathways, each with their own way of relaying information. G-protein coupled receptors can activate both phosphoinositol and cyclic AMP (cAMP) second messenger pathways. The phosphoinositol path is active when the receptor induces phospholipase C to hydrolyze the phospholipid, phosphatidylinositol biphosphate (PIP2), into two second messengers: diacylglycerol (DAG) and inositol triphosphate (IP3). DAG remains near the cell membrane and activates protein kinase C (PKC). IP3 translocates to the endoplasmic reticulum (ER) and becomes the opening ligand for calcium ion channels on the ER membrane- releasing calcium into the cytoplasm. In the cAMP pathway, the activated receptor induces adenylate cyclase to produce multiple copies of cAMP from nearby adenosine triphosphate (ATP) molecules. cAMP can stimulate protein kinase A (PKA), open calcium ion channels, and initiate the enzyme- Exchange-protein activated by cAMP (Epac). Similar to cAMP, is cyclic guanosine monophosphate (cGMP). cGMP is synthesized from guanosine triphosphate (GTP) molecules when guanylyl cyclase is activated. As a second messenger, cGMP induces protein kinase G

 Core: Cell Signaling

What are Lipids?

JoVE 10683

Lipids are a group of structurally and functionally diverse organic compounds that are insoluble in water. Certain classes of lipids, such as fats, phospholipids, and steroids are crucial to all living organisms. They function as structural components of cellular membranes, energy reservoirs, and signaling molecules.

Lipids are structurally and functionally diverse group of hydrocarbons. Hydrocarbons are chemical compounds that consist of carbon and hydrogen atoms. The carbon-carbon and carbon-hydrogen bonds are nonpolar, which means that the electrons between the atoms are shared equally. The individual nonpolar bonds impart an overall nonpolar characteristic to the hydrocarbon compound. Additionally, nonpolar compounds are hydrophobic, or “water-hating.” This means they do not form hydrogen bonds with water molecules, rendering them nearly insoluble in water. Depending on the chemical composition, lipids can be divided into different classes. The biologically important classes of lipids are fats, phospholipids, and steroids. The hydrocarbon backbone of fat has three carbon atoms. Each carbon carries a hydroxyl (–OH) group, making it glycerol. To form a fat, each of the hydroxyl groups of glycerol is linked to a fatty acid. A fatty acid is a long hydrocarbon chain with a carboxyl grou

 Core: Macromolecules

Macromolecules- Concept

JoVE 10590

Biomolecules

Organisms contain a wide variety of organic molecules with numerous functions which depend on the chemical structures and properties of these molecules. All organic molecules contain a carbon backbone and hydrogen atoms. The carbon atom is central in the formation of a vast variety of organic molecules ranging in size, shape and complexity; inorganic molecules on the other…

 Lab Bio

The Synapse

JoVE 10997

Neurons communicate with one another by passing on their electrical signals to other neurons. A synapse is the location where two neurons meet to exchange signals. At the synapse, the neuron that sends the signal is called the presynaptic cell, while the neuron that receives the message is called the postsynaptic cell. Note that most neurons can be both presynaptic and postsynaptic, as they both transmit and receive information. An electrical synapse is one type of synapse in which the pre- and postsynaptic cells are physically coupled by proteins called gap junctions. This allows electrical signals to be directly transmitted to the postsynaptic cell. One feature of these synapses is that they can transmit electrical signals extremely quickly—sometimes at a fraction of a millisecond—and do not require any energy input. This is often useful in circuits that are part of escape behaviors, such as that found in the crayfish that couples the sensation of a predator with the activation of the motor response. In contrast, transmission at chemical synapses is a stepwise process. When an action potential reaches the end of the axonal terminal, voltage-gated calcium channels open and allows calcium ions to enter. These ions trigger fusion of neurotransmitter-containing vesicles with the cellular membrane, releasing neurotransmitters into the small space b

 Core: Nervous System

Synaptic Signaling

JoVE 10717

Neurons communicate at synapses, or junctions, to excite or inhibit the activity of other neurons or target cells, such as muscles. Synapses may be chemical or electrical.

Most synapses are chemical. That means that an electrical impulse—or action potential—spurs the release of chemical messengers. These chemical messengers are also called neurotransmitters. The neuron sending the signal is called the presynaptic neuron. The neuron receiving the signal is the postsynaptic neuron. The presynaptic neuron fires an action potential that travels through its axon. The end of the axon, or axon terminal, contains neurotransmitter-filled vesicles. The action potential opens voltage-gated calcium ion channels in the axon terminal membrane. Ca2+ rapidly enters the presynaptic cell (due to the higher external Ca2+ concentration), enabling the vesicles to fuse with the terminal membrane and release neurotransmitters. The space between presynaptic and postsynaptic cells is called the synaptic cleft. Neurotransmitters released from the presynaptic cell rapidly populate the synaptic cleft and bind to receptors on the postsynaptic neuron. The binding of neurotransmitters instigates chemical changes in the postsynaptic neuron, such as opening or closing ion channels. This, in turn, alters the membrane potential of the postsynapti

 Core: Cell Signaling

Transformation of E. coli Cells Using an Adapted Calcium Chloride Procedure

JoVE 10515

Source: Natalia Martin1, Andrew J. Van Alst1, Rhiannon M. LeVeque1, and Victor J. DiRita1
1 Department of Microbiology and Molecular Genetics, Michigan State University


Bacteria have the ability to exchange genetic material (DeoxyriboNucleic Acid, DNA) in a process known as horizontal gene transfer. Incorporating exogenous DNA…

 Microbiology

G-protein Coupled Receptors

JoVE 10718

G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.

The G-protein or guanine nucleotide-binding protein, is a large heterotrimeric complex. Its three subunits are labeled alpha (α), beta (β), and gamma (γ). When the receptor is unbound or resting, the α-subunit binds a guanosine diphosphate molecule or GDP, and all three subunits are attached to the receptor. When a ligand binds the receptor, the α-subunit releases the GDP and binds a molecule of guanosine triphosphate (GTP). This action releases the α-GTP complex and the β-γ complex from the receptor. The α-GTP can move along the membrane to activate second messenger pathways such as cAMP. However there are different types of α-subunits and some are inhibitory, turning off cAMP. The β-γ complex may interact with potassium ion channels which release potassium (K+) into the extracellular space resulting in hyperpolarization of the cell membrane. This type of ligand-gated ion channel is called a G-prot

 Core: Cell Signaling

Tonicity in Animals

JoVE 10702

The tonicity of a solution determines if a cell gains or loses water in that solution. The tonicity depends on the permeability of the cell membrane for different solutes and the concentration of nonpenetrating solutes in the solution within and outside of the cell. If a semipermeable membrane hinders the passage of some solutes but allows water to follow its concentration gradient, water moves from the side with low osmolarity (i.e., less solute) to the side with higher osmolarity (i.e., higher solute concentration). Tonicity of the extracellular fluid determines the magnitude and direction of osmosis and results in three possible conditions: hypertonicity, hypotonicity, and isotonicity. In biology, the prefix “iso” means equal or being of equal measurements. When extracellular and intracellular fluid have an equal concentration of nonpenetrating solute inside and outside, the solution is isotonic. Isotonic solutions have no net movement of water. Water will still move in and out, just in equal proportions. Therefore, no change in cell volume occurs. The prefix “hypo” means lower or below. Whenever there is a low concentration of nonpenetrating solute and a high concentration of water outside relative to inside, the environment is hypotonic. Water will move into the cell, causing it to swell. In animal cells, the swelling ul

 Core: Membranes and Cellular Transport

Neuron Structure

JoVE 10842

Neurons are the main type of cell in the nervous system that generate and transmit electrochemical signals. They primarily communicate with each other using neurotransmitters at specific junctions called synapses. Neurons come in many shapes that often relate to their function, but most share three main structures: an axon and dendrites that extend out from a cell body.

The neuronal cell body—the soma— houses the nucleus and organelles vital to cellular function. Extending from the cell body are thin structures that are specialized for receiving and sending signals. Dendrites typically receive signals while the axon passes on the signals to other cells, such as other neurons or muscle cells. The point at which a neuron makes a connection to another cell is called a synapse. Neurons receive inputs primarily at postsynaptic terminals, which are frequently located on spines—small bumps protruding from the dendrites. These specialized structures contain receptors for neurotransmitters and other chemical signals. Dendrites are often highly branched, allowing some neurons to receive tens of thousands of inputs. Neurons most commonly receive signals at their dendrites, but they can also have synapses in other areas, such as the cell body. The signal received at the synapses travels down the dendrite to the soma, where the cell can proce

 Core: Nervous System

Endocrine Signaling

JoVE 10719

Endocrine cells produce hormones to communicate with remote target cells found in other organs. The hormone reaches these distant areas using the circulatory system. This exposes the whole organism to the hormone but only those cells expressing hormone receptors or target cells are affected. Thus, endocrine signaling induces slow responses from its target cells but these effects also last longer. There are two types of endocrine receptors: cell surface receptors and intracellular receptors. Cell surface receptors work similarly to other membrane bound receptors. Hormones, the ligand, bind to a hormone specific G-protein coupled receptor. This initiates conformational changes in the receptor, releasing a subunit of the G-protein. The protein activates second messengers which internalize the message by triggering signaling cascades and transcription factors. Many hormones work through cell surface receptors, including epinephrine, norepinephrine, insulin, prostaglandins, prolactin, and growth hormones. Steroid hormones, like testosterone, estrogen, and progesterone, transmit signals using intracellular receptors. These hormones are small hydrophobic molecules so they move directly past the outer cell membrane. Once inside, and if that cell is a target cell, the hormone binds to its receptor. Binding creates a conformational change in the receptor

 Core: Cell Signaling

Bacterial Transformation: The Heat Shock Method

JoVE 5059

Transformation is the process that occurs when a cell ingests foreign DNA from its surroundings. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the environment are called competent cells. In the…

 Basic Methods in Cellular and Molecular Biology

Yeast Signaling

JoVE 10714

Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes—cells that have a nucleus. Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule outside the yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases (enzymes that phosphorylate specific substrates) to activate or inactivate transcription factors that regulate gene expression. Many of the yeast intracellular signaling cascades have similar counterparts in Homo sapiens, making yeast a convenient model for studying intracellular signaling in humans. Yeasts are members of the fungus kingdom. They use signaling for various functions, especially for reproduction. Yeasts can undergo “sexual” reproduction using mating pheromones, which are peptides—short chains of amino acids. Yeast colonies consist of both diploid and haploid cells. Both types of cells can undergo mitosis, but only diploid cells can undergo meiosis. When diploid cells undergo meiosis, the four resulting haploid cells, called spores, are not identical. In fact, the division of one diploid cell into four spores creates two “sexes” of yeast cells, each two cells of the type MAT-a and MAT-alpha. MAT-a cells secrete mating

 Core: Cell Signaling

FM Dyes in Vesicle Recycling

JoVE 5648

FM dyes are a class of fluorescent molecules that has found important use in studying the vesicle recycling process. By virtue of a chemical structure, these molecules can insert themselves into the outer leaflet of phospholipid bilayer membranes. After membrane insertion, they are internalized into the cell via endocytosed vesicles, and released when these vesicles…

 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

DNA Isolation and Restriction Enzyme Analysis- Concept

JoVE 10628

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and…

 Lab Bio

Bacterial Transformation- Concept

JoVE 10573

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance…

 Lab Bio

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 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

Retrovirus Life Cycles

JoVE 10825

Retroviruses have a single-stranded RNA genome that undergoes a special form of replication. Once the retrovirus has entered the host cell, an enzyme called reverse transcriptase synthesizes double-stranded DNA from the retroviral RNA genome. This DNA copy of the genome is then integrated into the host’s genome inside the nucleus via an enzyme called integrase. Consequently, the retroviral genome is transcribed into RNA whenever the host’s genome is transcribed, allowing the retrovirus to replicate. New retroviral RNA is transported to the cytoplasm, where it is translated into proteins that assemble new retroviruses. Particular drugs have been developed to fight retroviral infections. These drugs target specific aspects of the life cycle. One class of antiretroviral drugs, fusion inhibitors, prevents the entry of the retrovirus into the host cell by inhibiting the fusion of the retrovirus with the host cell membrane. Another class of antiretrovirals, reverse transcriptase inhibitors, inhibits the reverse transcriptase enzymes that make DNA copies of the retroviral RNA genome. Reverse transcriptase inhibitors are competitive inhibitors; during the process of reverse transcription, the drug molecules are incorporated into the growing DNA strand instead of the usual DNA bases. Once incorporated, the drug molecules block further progress by the r

 Core: Viruses

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

Spermatogenesis

JoVE 10905

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of seminiferous tubules. These spermatogonial stem cells divide asymmetrically to give rise to additional stem cells (meaning that these structures “self-renew”), as well as sperm progenitors, called spermatocytes. Importantly, this method of asymmetric mitotic division maintains a population of spermatogonial stem cells in the male reproductive tract, ensuring that sperm will continue to be produced throughout a man’s lifespan. As spermatogenesis proceeds, spermatocytes embark on meiosis, and each ultimately divides to form four sperm—each with only 23 chromosomes— that are expelled into the male reproductive tract. Interestingly, this is in contrast to oogenesis in women, during which only a single egg is generated for every progenitor cell. At the end of spermatogenesis, sperm demonstrate their characteristic shape: a “head” harboring minimal cytoplasm and a highly condensed nucleus, as well as a motile tail (flagellum). They are small cells, with no organelles such as ribosomes, ER or Golgi, but do have many mitochondria around the flagellum for power. Just below the head is the acrosomal vesicle which contains hydrolytic enzymes to penetrate the egg outer coat—th

 Core: Reproduction and Development

Energy-requiring Steps of Glycolysis

JoVE 10738

Glucose is the source of nearly all energy used by organisms. The first step of converting glucose into usable energy is called glycolysis. Glycolysis occurs in the cytosol of the cell over two phases: an energy-requiring phase and an energy-releasing phase. Over the first three steps, glucose is converted into different forms and attaches to two phosphate groups donated by two ATP molecules, resulting in an unstable sugar. In the next two stages, the unstable sugar splits into two sugar isomers which are either converted or used directly in the next phase of glycolysis. First, glucose receives a phosphate group from ATP converting it into a more reactive form (glucose 6-phosphate). Because glucose attached to the negatively-charged phosphate cannot cross the hydrophobic cell membrane, the addition of a phosphate group also traps glucose inside the cell. Next, the more reactive form of glucose is converted into one of its isomers, fructose 6-phosphate, which is required for subsequent energy-requiring steps of glycolysis. Fructose 6-phosphate then receives a phosphate group from a second ATP molecule. This converts fructose 6-phosphate into fructose 1,6-bisphosphate, an unstable sugar. This unstable sugar splits into two distinct three-carbon sugar isomers, glyceraldehyde 3-phosphate and DHAP. Glyceraldehyde 3-phosphate can be directly use

 Core: Cellular Respiration

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

Intracellular Hormone Receptors

JoVE 10876

Lipid-soluble hormones diffuse across the plasma and nuclear membrane of target cells to bind to their specific intracellular receptors. These receptors act as transcription factors that regulate gene expression and protein synthesis in the target cell

Based on their mode of action, intracellular hormone receptors are classified as Type I or Type II receptors. Type I receptors, including steroid hormone receptors such as the androgen receptor, are present in the cytoplasm. Hormone binding transports the hormone-receptor complex to the nucleus, where it binds to regulatory DNA sequences called hormone response elements and activates gene transcription. Type II receptors, such as the thyroid hormone receptor, are bound to their DNA response elements within the nucleus even in the absence of hormone. In this state, the receptor acts as an active repressor of transcription. However, upon hormone binding, the receptor-hormone complex activates transcription of thyroid hormone-inducible genes.

 Core: Endocrine System

In-vitro Mutagenesis

JoVE 10813

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

Genes can be randomly knocked out, or specific genes can be targeted. To knock out a particular gene, an engineered piece of DNA called a targeting vector is used to replace the normal gene, thereby inactivating it. Targeting vectors have sequences on each end that are identical—or homologous— to the sequences flanking each side of the gene of interest. These homologous sequences allow the targeting vector to replace the gene through homologous recombination—a process that occurs naturally between DNA with similar sequences during meiosis. The targeting vector is introduced into mouse embryonic stem cells in culture, using methods such as electroporation—use of electric pulses to temporarily create pores in the cell membrane. Typically, to identify cells where the vector has properly replaced the gene, it is designed to include a positive selection marker—such as the gene for neomycin resistance (NeoR)—between the homologous regions; and a negative selection marker—such as th

 Core: Biotechnology

What are Proteins?

JoVE 10677

Proteins are chains of amino acids that are connected by peptide bonds and folded into a 3-dimensional structure. The side chains of individual amino acid residues determine the interactions among amino acid residues, and ultimately the folding of the protein. Depending on the length and structural complexity, chains of amino acid residues are classified as oligopeptides, polypeptides, or proteins. An amino acid is a molecule that contains a carboxyl (–COOH) and an amino group (–NH2) attached to the same carbon atom, the ⍺-carbon. The identity of the amino acid is determined by its side chain or side residue, often called the R-group. The simplest amino acid is glycine, where the residue is a single hydrogen atom. Other amino acids carry more complex side chains. The side chain determines the chemical properties of the amino acid. For example, it may attract or repel water (hydrophilic or hydrophobic), carry a negative charge (acidic), or form hydrogen bonds (polar). Of all known amino acids, only 21 are used to create proteins in eukaryotes (the genetic code encodes only 20 of these). Amino acids are abbreviated using a three letter (e.g., Gly, Val, Pro) or one letter code (e.g., G, V, P). The linear chain of amino acid residues forms the backbone of the protein. The free amino group at one end is called the N-terminus, while t

 Core: Macromolecules

Physiology of the Circulatory System- Concept

JoVE 10625

Homeostasis

Conditions in the external environment of an organism can change rapidly and drastically. To survive, organisms must maintain a fairly constant internal environment, which involves continuous regulation of temperature, pH, and other factors. This balanced state is known as homeostasis, which describes the processes by which organisms maintain their optimal internal…

 Lab Bio

An Introduction to Transfection

JoVE 5068

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for…

 Basic Methods in Cellular and Molecular Biology

Ionic Bonds

JoVE 10665

When atoms gain or lose electrons to achieve a more stable electron configuration they form ions. Ionic bonds are electrostatic attractions between ions with opposite charges. Ionic compounds are rigid and brittle when solid and may dissociate into their constituent ions in water. Covalent compounds, by contrast, remain intact unless a chemical reaction breaks them.

Ionic bonds are reversible electrostatic interactions between ions with opposing charges. Elements that are the most reactive (i.e., have a higher tendency to undergo chemical reactions) include those that only have one valence electron, (e.g., potassium) and those that need one more valence electron (e.g., chlorine). Ions that lose electrons have a positive charge and are referred to as cations. Ions that gain electrons have a negative charge and are called anions. Cations and anions combine in ratios that result in a net charge of 0 for the compound they form. For example, the compound potassium chloride (KCl) contains one chloride ion for each potassium ion, because the charge of potassium is +1 and the charge of chloride is -1. The compound magnesium chloride (MgCl2) contains two chloride ions for each magnesium ion because magnesium’s charge is +2. The electrostatic forces holding ionic compounds together are strong when the compounds are in solid form. Since t

 Core: Chemistry of Life

Using Diffusion Tensor Imaging in Traumatic Brain Injury

JoVE 10276

Source: Laboratories of Jonas T. Kaplan and Sarah I. Gimbel—University of Southern California


Traditional brain imaging techniques using MRI are very good at visualizing the gross structures of the brain. A structural brain image made with MRI provides high contrast of the borders between gray and white matter, and information about…

 Neuropsychology
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