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

Em temperaturas fisiológicas, uma bicamada lipídica atua como um fluido bidimensional que permite a difusão de proteínas, lipídios e outros componentes da membrana.
A difusão lateral de proteínas é um movimento lateral dentro da membrana.
A matriz extracelular pode interagir com o domínio extracelular de uma proteína e interferir em seu movimento, ou intracelularmente, o citoesqueleto de actina e as proteínas transmembranares associadas podem formar cercas, resultando na criação de compartimentos específicos conhecidos como currais.
Essas cercas do citoesqueleto obstruem o livre movimento de proteínas de um curral para outro. No entanto, a renovação da actina pode criar lacunas temporárias na cerca que podem permitir que as proteínas se movam de um curral para outro.
A taxa de difusão lateral de proteínas pode ser determinada pela recuperação de fluorescência após fotobranqueamento ou FRAP. Nesta técnica, as proteínas integrais da membrana são marcadas com sondas fluorescentes.
Quando uma célula imobilizada é irradiada com um feixe de laser, a sonda fluorescente torna-se fotobranqueada, resultando em uma área circular não fluorescente. A fluorescência perdida se recupera gradualmente à medida que as proteínas não branqueadas se difundem para a área branqueada.

difusão de proteínas na membrana

As proteínas apresentam difusão rotacional e também lateral através da membrana. A difusão lateral de proteínas foi confirmada através do experimento de fusão celular, onde células de camundongo e humanas foram fundidas, resultando em células híbridas. Quando as células humanas e de camundongo se fundiram, as proteínas específicas da membrana nas células humanas e de camundongo foram marcadas com marcadores fluorescentes vermelhos e verdes, respectivamente. Inicialmente, a fluorescência vermelha e verde estava localizada no seu respectivo hemisfério da célula. Com o passar do tempo, as proteínas marcadas com marcadores fluorescentes vermelhos e verdes começaram a se mover lateralmente e foram completamente misturadas em toda a superfície celular em 40 minutos. Essa experiência confirmou que as proteínas da membrana podem apresentar difusão lateral. A técnica de recuperação de fluorescência após fotodegradação (FRAP) pode ser usada para determinar a taxa de difusão de proteínas da membrana. Nessa técnica, a proteína-alvo é marcada por um anticorpo marcado com fluoróforo que é específico da proteína. Alternativamente, a engenharia genética também pode ser usada para produzir a proteína-alvo fundida com a proteína verde fluorescente. O feixe de laser é usado para criar a área branqueada não fluorescente. Se ocorrer uma recuperação de fluorescência de 40%, considera-se que cerca de 40% das proteínas da membrana são difusíveis lateralmente. A taxa de difusão pode então ser calculada utilizando o tempo necessário para a recuperação da fluorescência na área branqueada e, por sua vez, pode ser utilizado para determinar o coeficiente de difusão. Dependendo da localização e das estruturas da proteína, cada proteína possui um coeficiente de difusão diferente. Os estudos de FRAP mostraram que nem todas as proteínas da membrana são igualmente móveis, e a taxa de difusão das proteínas da membrana é mais lenta do que a esperada para a bicamada lipídica pura. A taxa de difusão das proteínas através da membrana é inferior à taxa de difusão dos lipídios da membrana. Por exemplo, para atravessar o comprimento de 20 micrômetros de uma célula eucariótica, os lipídios da membrana levam cerca de 20 segundos, enquanto as proteínas da membrana podem levar de 600 a 3.600 segundos. Além do FRAP, outras técnicas avançadas como 1) rastreamento de partículas únicas, 2) utilização de pinça óptica para estudo das barreiras de membrana e 3) proteínas geneticamente modificadas para se observar o papel dos domínios proteicos citoplasmáticos e exoplasmáticos na difusão lateral foram utilizadas para explorar vários aspectos da difusão proteica na membrana.

Difusão de proteínas na membrana

As proteínas mostram difusão rotacional e lateral através da membrana. A difusão lateral de proteínas foi confirmada através da fusão celular ...

Education

JoVE Core

Cell Biology

Biologia Celular

Unit 1

Unidade 1

Membrane Structure and Components

Estrutura e Componentes da Membrana

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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 - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

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Co-activators and Co-repressors

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Proteins: From Genes to Degradation

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9-12.B.1.2

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9-12.B.1.2.a

How Membrane Proteins Move Side to Side

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