Name: how membrane proteins move side to side
Uploaded: 2023-04-30T09:16:22.000Z
Duration: 1 min 24 s
Channel: JoVE

K12 Standards

Alabama Course of Study Science

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.4.a

Protein Diffusion in the Membrane

A temperature fisiologiche, un doppio strato lipidico agisce come un fluido bidimensionale che consente la diffusione di proteine, lipidi e altri componenti della membrana.
La diffusione laterale delle proteine è il movimento laterale all'interno della membrana.
La matrice extracellulare può interagire con il dominio extracellulare di una proteina e interferire con il suo movimento, oppure intracellulare, il citoscheletro di actina e le proteine transmembrana associate possono formare recinzioni con conseguente creazione di compartimenti specifici noti come recinti.
Queste recinzioni citoscheletriche ostruiscono il libero movimento delle proteine da un recinto all'altro. Tuttavia, il ricambio di actina può creare spazi temporanei nella recinzione che possono consentire alle proteine di spostarsi da un recinto all'altro.
La velocità di diffusione laterale delle proteine può essere determinata dal recupero della fluorescenza dopo fotobleaching o FRAP. In questa tecnica, le proteine integrali di membrana sono marcate con sonde fluorescenti.
Quando una cellula immobilizzata viene irradiata con un raggio laser, la sonda fluorescente diventa fotosbiancata, risultando in un'area circolare non fluorescente. La fluorescenza persa si riprende gradualmente man mano che le proteine non sbiancate si diffondono nell'area sbiancata.

diffusione delle proteine nella membrana

Le proteine presentano una diffusione rotazionale e laterale attraverso la membrana. La diffusione laterale delle proteine è stata confermata attraverso l'esperimento di fusione cellulare in cui cellule di topo e umane sono state fuse, dando origine a cellule ibride. Quando le cellule umane e di topo si sono fuse, le specifiche proteine di membrana nelle cellule umane e di topo sono state contrassegnate rispettivamente con marcatori fluorescenti rossi e verdi. Inizialmente, la fluorescenza rossa e verde era localizzata sul rispettivo emisfero della cellula. Con il passare del tempo, le proteine contrassegnate con marcatori fluorescenti rossi e verdi hanno iniziato a muoversi lateralmente e si sono completamente mescolate su tutta la superficie cellulare in 40 minuti. Questo esperimento ha confermato che le proteine di membrana possono mostrare diffusione laterale.

La tecnica di recupero della fluorescenza dopo il fotosbiancamento (FRAP) può essere utilizzata per determinare la velocità di diffusione delle proteine di membrana. In questa tecnica, la proteina bersaglio è contrassegnata da un anticorpo marcato con fluoroforo specifico per la proteina. In alternativa, è possibile utilizzare anche l'ingegneria genetica per produrre la proteina bersaglio fusa con la proteina fluorescente verde. Il raggio laser viene utilizzato per creare l'area sbiancata non fluorescente. Se si verifica un recupero della fluorescenza del 40%, si considera che circa il 40% delle proteine di membrana sono diffusibili lateralmente. La velocità di diffusione può quindi essere calcolata utilizzando il tempo richiesto per il recupero della fluorescenza nell'area sbiancata, che a sua volta può essere utilizzato per determinare il coefficiente di diffusione. A seconda della posizione e della struttura della proteina, ciascuna proteina ha un coefficiente di diffusione diverso.

Gli studi del FRAP hanno dimostrato che non tutte le proteine di membrana sono ugualmente mobili e che la velocità di diffusione delle proteine di membrana è più lenta di quella prevista dal doppio strato lipidico puro. La velocità di diffusione delle proteine attraverso la membrana è inferiore alla velocità di diffusione dei lipidi di membrana. Ad esempio, per attraversare la lunghezza di 20 micrometri di una cellula eucariotica, i lipidi di membrana impiegano circa 20 secondi, mentre le proteine di membrana possono impiegare fino a 600-3600 secondi. Oltre al FRAP, sono state utilizzate altre tecniche avanzate per esplorare vari aspetti della diffusione proteica nella membrana, tra cui 1) tracciamento di singole particelle; 2) l'uso di pinzette ottiche per studiare le barriere di membrana; e 3) proteine geneticamente modificate per vedere il ruolo dei domini proteici citoplasmatici ed esoplasmici nella diffusione laterale.

Diffusione delle proteine nella membrana

Le proteine mostrano una diffusione rotazionale e laterale attraverso la membrana. La diffusione laterale delle proteine è stata confermata attraverso la fusione cellulare ...

Education

Didattica

JoVE Core

Cell Biology

Biologia cellulare

Unit 1

Unità 1

Membrane Structure and Components

Struttura e componenti della membrana

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

how membrane proteins move side to side

Membrane proteins can move across the cell surface by lateral diffusion, and they can also rotate in place. Scientists confirmed lateral movement with a cell fusion experiment. Mouse and human cells were fused to make hybrid cells. The membrane proteins on the two cells were tagged with red and green fluorescent markers. At first, each color stayed on its own side of the fused cell. After 40 minutes, the colors mixed across the whole cell surface, showing that the proteins had moved laterally.

FRAP, or fluorescence recovery after photobleaching, is another way to measure membrane protein diffusion. In this method, the target protein is labeled with a fluorophore-linked antibody that binds only to that protein. A genetically engineered protein fused to green fluorescent protein can also be used. A laser then creates a bleached, non-fluorescent area. If 40% of the fluorescence returns, about 40% of the proteins in that area are considered laterally diffusible.

The time needed for recovery helps scientists calculate the diffusion rate and the diffusion coefficient. Different proteins have different diffusion coefficients because their location and structure are not the same. FRAP studies also showed that membrane proteins move more slowly than expected from the pure lipid bilayer. Their diffusion is slower than membrane lipids. For example, lipids may cross the 20-micrometer length of a eukaryotic cell in about 20 seconds, while membrane proteins may take 600 to 3600 seconds.

Researchers also use other advanced methods to study membrane protein movement. These include single-particle tracking, optical tweezers to study membrane barriers, and genetically modified proteins that help show how cytoplasmic and exoplasmic domains affect lateral diffusion.

How Membrane Proteins Move Side to Side

what are membranes?

Membrane Receptors and Cell Signals

The plasma membrane sets the outer boundary of a cell and helps control how the cell interacts with its environment. It lets some substances enter, keeps others out, and releases some materials in controlled amounts. The membrane also needs to stay flexible so cells such as red and white blood cells can change shape as they move through narrow capillaries. The membrane surface also carries markers that help cells recognize one another. This recognition is important during early development,...

membrane fluidity

How Cell Membranes Stay Flexible

Cell membranes stay flexible because of the fluid mosaic model. The plasma membrane is a mosaic of phospholipids, cholesterol, proteins, and carbohydrates. These parts are separate but loosely attached, so the membrane keeps a fluid character. The membrane is still fairly rigid, but it can flow and self-seal after a very fine needle passes through it. Phospholipid tails also affect membrane fluidity. Saturated fatty acids in phospholipids have straight tails because they are packed with...

fluid mosaic model

Fluid Mosaic Model of the Membrane

The fluid mosaic model explains the structure and function of the plasma membrane. Scientists first identified the plasma membrane in the 1890s, and by 1915 they had identified its main chemical parts: lipids and proteins. Early electron micrographs showed a railroad track pattern, which helped lead to the first widely accepted membrane model in 1935.

membrane lipids

Membrane Lipids in Cells and Tissues

Membrane lipids give biological membranes their structure and help set their properties. In mammalian cells, lipids make up about 50% of the membrane on average. The amount can be much lower in the inner mitochondrial membrane or much higher in the myelin sheath around nerve cells. Several phospholipids are common in mammalian membranes. These include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. At physiological pH, phosphatidylserine carries a negative...

asymmetric lipid bilayer

Membrane Lipid Unevenness

Biological membranes have an uneven mix of lipids between the inner and outer layers. This membrane asymmetry gives each side a different chemical makeup and supports different cell functions. Evidence for this uneven layout came from treating an erythrocyte membrane with phospholipase. Phospholipase is an enzyme that breaks lipids into fatty acids and hydrophilic groups. It acted only on the outer layer, while the inner layer stayed intact. The treatment degraded about 80% of total membrane...

membrane asymmetry regulating transporters

membrane-asymmetry-regulating-transporters

Membrane Lipid Flipping and Transport

Membrane lipid flipping and transport help cells keep an uneven, or asymmetric, distribution of phospholipids across the membrane. This balance is controlled by flippase, floppase, and scramblase enzymes, which move lipids between the two membrane leaflets. Flippases move specific phospholipids from the outer leaflet to the inner leaflet. In eukaryotes, these enzymes are type-IV P-type ATPases, also called P4-ATPases. They are membrane pumps that use ATP and contain one transmembrane domain...

membrane carbohydrates

Membrane Sugars and Cell Identity

Membrane carbohydrates help give cells their surface identity and support plasma membrane function. The plasma membrane is a dynamic barrier made of lipids, proteins, and carbohydrates. These sugar chains have unique structural and chemical traits that help the membrane work properly for cell growth and survival. Membrane carbohydrates do not have a hydrophobic region, so they are found only on the outer surface of the cell. Sugar addition, or glycosylation, of proteins occurs in the lumens of...

membrane proteins

Facilitated Transport Across Cell Membranes

Facilitated transport uses membrane proteins to help materials cross the plasma membrane. These integral transmembrane proteins act as channels or carriers. They move selected substances through the membrane more easily than the lipid layer alone would allow. Channel proteins provide a hydrophilic, or water-friendly, path through the membrane. Their core forms a hydrated opening that lets solutes pass between the inside and outside fluids of the cell. This helps polar compounds avoid the...

lipids as anchors

Lipid Anchors in Membrane Proteins

Lipid anchors help attach proteins to the plasma membrane. In eukaryotic cells, the main lipid anchors are prenyl groups, fatty acyl groups, and glycosylphosphatidylinositol, or GPI, groups. Prenyl and fatty acyl groups anchor proteins on the cytosolic side of the membrane. GPI anchors proteins on the extracellular side. Prenylated proteins often have a CaaX motif at the carboxy terminus. In this sequence, C means cysteine, a means any aliphatic amino acid, and X means any other amino acid...

single-pass transmembrane proteins

How Membrane Proteins Span Cell Membranes

Membrane proteins help cells communicate, send signals, stick to other cells, and move molecules across the membrane. Integral membrane proteins are tightly linked to the cell membrane and carry out these jobs. Some integral membrane proteins sit only in one membrane layer. Fatty acid amide hydrolase is one example, and it is found on the cytoplasmic side of the membrane monolayer. Other integral membrane proteins are transmembrane proteins, which span across the membrane. These proteins have...

multi-pass transmembrane proteins and &#946;-barrels

multi-pass-transmembrane-proteins-and-barrels

Membrane-Spanning Helices and β-Barrels

Membrane-spanning proteins can cross the cell membrane more than once. Their transmembrane chain usually folds into either an alpha-helix or a beta-strand. Alpha-helix multi-pass proteins are common, while beta-strand proteins are found mainly in gram-negative bacteria, mitochondria, and chloroplasts. Some multi-pass alpha-helix proteins are G-protein-linked receptors, or GPCRs, and bacteriorhodopsin. GPCRs have seven transmembrane alpha-helices. Each receptor also has its own extracellular...

detergent purification of membrane proteins

detergent-purification-of-membrane-proteins

How Detergents Solubilize Membrane Proteins

Detergents help solubilize membrane proteins so they can be purified from the lipid bilayer. Their hydrophobic region can replace membrane phospholipids and keep integral membrane proteins in solution. A detergent works well only when its monomers reach the critical micelle concentration, or CMC. At that point, the molecules assemble into micelles, which are small clusters of detergent. Above the CMC, free detergent monomers stay in equilibrium with the micelles. The CMC varies with the...

protein diffusion in the membrane

Membrane proteins can move across the cell surface by lateral diffusion, and they can also rotate in place. Scientists confirmed lateral movement with a cell fusion experiment. Mouse and human cells were fused to make hybrid cells. The membrane proteins on the two cells were tagged with red and green fluorescent markers. At first, each color stayed on its own side of the fused cell. After 40 minutes, the colors mixed across the whole cell surface, showing that the proteins had moved laterally. <br...

membrane domains

Lipid Rafts and Sperm Membrane Regions

Membrane domains organize specific lipids and proteins in defined parts of a cell membrane. This arrangement supports cell signaling, adhesion, and other important cellular processes. These domains can vary in size, composition, function, and how long they last. Some membrane domains are built from proteins. The membrane can contain distinct protein groups that carry out a cell’s specific functions. A clear example is the plasma membrane of a human sperm cell, which has different proteins in...

mechanisms of membrane domain formation

How Cell Membranes Form Domains

Cell membranes form domains when lipids and proteins gather into distinct regions. These regions, or membrane domains, are shaped by different physical properties that help some molecules stay together while others move apart. Some domains form through protein-protein interactions. Others depend on specific lipids, such as sphingolipids and sterols. Large proteins, including bacteriorhodopsin, can also cluster and create visible membrane domains. Membrane proteins can be held in place by...

mechanisms of membrane-bending

How Proteins Shape Cell Membranes

Cell membranes can bend into many shapes because they are flexible and made of a fluid mosaic. That bending can be temporary, as in vesicles, or long-lasting, as in microvilli. Cells control the size, place, and time of membrane curvature. Membrane bending can start inside the membrane itself or from proteins attaching to it. Changes in lipid makeup can create curvature, and proteins can help by making the membrane asymmetric or by clustering certain lipids. The shape of integral proteins also...

key terms and concepts

Learn how membrane proteins move laterally in the membrane, how cell fusion and FRAP measure diffusion, and why proteins move slower than lipids.

How Membrane Proteins Move Side to Side | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

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9-12.B.1.1

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9-12.B.1.2

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a. Use a model to describe the structure and sequence of DNA, including nucleotide structure, base pairing, and the structure of the helix.

9-12.B.1.2.a

How Membrane Proteins Move Side to Side

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