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

在生理温度下，脂质双分子层表现为一种二维流体，允许蛋白质、脂质及其他膜组分在其内扩散。
蛋白质的侧向扩散是指其在膜内的横向移动。
细胞外基质可与蛋白质的细胞外结构域相互作用，并干扰其运动；而在细胞内，肌动蛋白细胞骨架及其相关的跨膜蛋白可形成围栏，从而产生称为“围栏区”（corrals）的特定区域。
这些细胞骨架栅栏会阻碍蛋白质从一个围栏区域自由移动到另一个区域。然而，肌动蛋白的周转可暂时形成栅栏中的间隙，从而使蛋白质得以在不同围栏区域之间移动。
蛋白质的侧向扩散速率可通过光漂白后荧光恢复（FRAP）来测定。该技术中，整合膜蛋白使用荧光探针进行标记。
当固定细胞被激光束照射时，荧光探针会发生光漂白，从而形成一个圆形的非荧光区域。随着未漂白的蛋白质扩散至漂白区域，荧光逐渐恢复。

蛋白质在膜中的扩散

蛋白质在膜上既有旋转扩散，又有横向扩散。蛋白质的横向扩散是通过细胞融合实验来得到证实的，将小鼠细胞和人类细胞进行融合，便能够产生杂交细胞。当人类细胞和小鼠细胞融合后，人类细胞和小鼠细胞上的特定膜蛋白会分别被标记为红色和绿色荧光标记。最初，红色和绿色荧光分别位于细胞的半球上。随着时间的推移，标有红色和绿色荧光标记的蛋白质开始发生横向移动，并在 40 分钟后完全混合在整个细胞表面。该实验证实了膜蛋白可以进行横向扩散。

光漂白后荧光恢复（FRAP）技术能够用来测定膜蛋白的扩散速率。在这种技术中，目标蛋白质会被一种对蛋白质具有特异性的荧光团标记的抗体进行标记。另外，也可以利用基因工程技术来生产与绿色荧光蛋白融合的目标蛋白。激光束能够用来产生无荧光的漂白区域。如果有 40% 的荧光恢复，则可以认为大约有 40% 的膜蛋白是能够进行横向扩散的。然后可以利用漂白区域中荧光恢复所需的时间来计算扩散速率，进而能够确定扩散系数。根据蛋白质的位置和结构可以得知，每种蛋白质都有着不同的扩散系数。

光漂白后荧光恢复的研究表明了，并非所有的膜蛋白都具有同等的流动性，并且膜蛋白的扩散速率会比纯脂质双分子层的预期扩散速率要慢。蛋白质在整个膜上的扩散速率低于膜脂的扩散速率。例如，要穿过 20 微米长的真核细胞，那么膜脂大约需要 20 秒，而膜蛋白则可能需要长达 600-3600 秒。除了光漂白后荧光恢复之外，其他的先进技术例如：1）单粒子跟踪；2）使用光镊来研究膜屏障；3）转基因蛋白质，使用这些技术能够观察细胞质和外质蛋白质结构域在横向扩散中的作用，同时也被用来探索蛋白质在膜中扩散的各个方面。

蛋白质在膜上表现出旋转扩散以及侧向扩散。通过细胞融合实验已证实蛋白质的侧向扩散。

Education

教育

JoVE Core

Cell Biology

细胞生物学

Unit 1

第一单元

Membrane Structure and Components

膜结构与组成

KEY TERMS AND CONCEPTS

关键术语与概念

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

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9-12.B.1.2.a

How Membrane Proteins Move Side to Side

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