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Из Википедии, бесплатной энциклопедии
  (Перенаправлено с липидных бислоев )
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Это поперечное сечение жидкого липидного бислоя полностью состоит из фосфатидилхолина .
Три основные структуры фосфолипидов образуются в растворе; липосомы (замкнутое бислой), мицелла и бислой.

Липидный бислой (или фосфолипидный бислой ) представляет собой тонкую полярная мембрана изготовлена из двух слоев липидных молекул . Эти мембраны представляют собой плоские листы, которые образуют непрерывный барьер вокруг всех клеток . В клеточных мембранах практически всех организмов и многих вирусов сделаны из липидного бислоя, равно как и ядерная оболочка , окружающая ядро клетки и мембраны этих мембраносвязанных органелл в клетке. Липидный бислой - это барьер, который удерживает ионы , белки.и другие молекулы там, где они необходимы, и предотвращает их распространение в области, где они не должны быть. Липидные бислои идеально подходят для этой роли, даже несмотря на то, что их ширина составляет всего несколько нанометров [1], поскольку они непроницаемы для большинства водорастворимых ( гидрофильных ) молекул. Бислои особенно непроницаемы для ионов, что позволяет клеткам регулировать концентрацию солей и pH , транспортируя ионы через свои мембраны с помощью белков, называемых ионными насосами .

Биологические бислои обычно состоят из амфифильных фосфолипидов, которые имеют гидрофильную фосфатную головку и гидрофобный хвост, состоящий из двух цепей жирных кислот. Фосфолипиды с определенными головными группами могут изменять химический состав поверхности бислоя и могут, например, служить сигналами, а также «якорями» для других молекул в мембранах клеток. [2] Так же, как головы, хвосты липидов также могут влиять на свойства мембран, например, определяя фазу бислоя. Двухслойный слой может принимать твердое гелевое фазовое состояние при более низких температурах, но претерпевать фазовый переход в жидкое состояние.при более высоких температурах, и химические свойства липидных хвостов влияют на то, при какой температуре это происходит. Упаковка липидов внутри бислоя также влияет на его механические свойства, включая его сопротивление растяжению и изгибу. Многие из этих свойств были изучены с использованием искусственных «модельных» бислоев, созданных в лаборатории. Везикулы , образованные модельными двойными слоями, также используются в клинической практике для доставки лекарств.

Биологические мембраны обычно включают несколько типов молекул, кроме фосфолипидов. Особенно важным примером в клетках животных является холестерин , который помогает укрепить бислой и снизить его проницаемость. Холестерин также помогает регулировать активность некоторых интегральных мембранных белков . Интегральные мембранные белки функционируют, когда включены в липидный бислой, и они плотно удерживаются на липидном бислое с помощью кольцевой липидной оболочки.. Поскольку бислои определяют границы клетки и ее компартментов, эти мембранные белки участвуют во многих внутри- и межклеточных процессах передачи сигналов. Определенные виды мембранных белков участвуют в процессе слияния двух бислоев вместе. Это слияние позволяет соединение двух различных структур , как и в реакции акроса во время оплодотворения в качестве яйца с помощью спермы или попаданий в вирусе в клетку. Поскольку липидные бислои хрупки и невидимы в традиционный микроскоп, их сложно изучать. Эксперименты с бислоями часто требуют передовых методов, таких как электронная микроскопия и атомно-силовая микроскопия .

Структура и организация [ править ]

Когда фосфолипиды подвергаются воздействию воды, они сами собираются в двухслойный лист с гидрофобными хвостами, направленными к центру листа. Такое расположение приводит к двум «листочкам», каждый из которых представляет собой отдельный молекулярный слой. Центр этого бислоя почти не содержит воды и исключает молекулы, такие как сахара или соли, которые растворяются в воде. Процесс сборки управляется взаимодействиями между гидрофобными молекулами (также называемый гидрофобным эффектом ). Увеличение взаимодействий между гидрофобными молекулами (вызывая кластеризацию гидрофобных областей) позволяет молекулам воды более свободно связываться друг с другом, увеличивая энтропию системы. Этот сложный процесс включает нековалентные взаимодействия, такие каксилы Ван-дер-Ваальса , электростатические и водородные связи .

Анализ поперечного сечения [ править ]

Схематический профиль поперечного сечения типичного липидного бислоя. Есть три различных области: полностью гидратированные головные группы, полностью дегидратированное алкановое ядро ​​и короткая промежуточная область с частичной гидратацией. Хотя головные группы нейтральны, они обладают значительными дипольными моментами, которые влияют на расположение молекул. [3]

Липидный бислой очень тонкий по сравнению с его боковыми размерами. Если бы типичную клетку млекопитающего (диаметр ~ 10 микрометров) увеличить до размера арбуза (~ 1 фут / 30 см), липидный бислой, составляющий плазматическую мембрану, был бы толщиной примерно с лист офисной бумаги. Несмотря на толщину всего в несколько нанометров, бислой состоит из нескольких различных химических областей в поперечном сечении. Эти области и их взаимодействия с окружающей водой были охарактеризованы в течение последних нескольких десятилетий с помощью рентгеновской рефлектометрии , [4] рассеяния нейтронов [5] и методов ядерного магнитного резонанса .

Первая область по обе стороны от бислоя - это гидрофильная головная группа. Эта часть мембраны полностью гидратирована и обычно имеет толщину около 0,8-0,9 нм. В фосфолипидных бислоях фосфатная группа расположена внутри этой гидратированной области, примерно на 0,5 нм за пределами гидрофобного ядра. [6] В некоторых случаях гидратированная область может простираться намного дальше, например, в липидах с большим количеством белка или длинной сахарной цепью, привитой к голове. Одним из распространенных примеров такой модификации в природе является липополисахаридное покрытие на внешней мембране бактерий [7], которое помогает удерживать водный слой вокруг бактерии для предотвращения обезвоживания.

ПЭМ- изображение бактерии. Пушистый вид снаружи обусловлен слоем длинноцепочечных сахаров, прикрепленных к клеточной мембране. Это покрытие помогает удерживать воду и предотвращать обезвоживание бактерий.

Рядом с гидратированной областью находится промежуточная область, которая лишь частично гидратирована. Этот пограничный слой имеет толщину примерно 0,3 нм. На этом коротком расстоянии концентрация воды падает с 2M на стороне головной группы до почти нуля на стороне хвоста (сердцевины). [8] [9] Гидрофобное ядро ​​бислоя обычно имеет толщину 3-4 нм, но это значение зависит от длины цепи и химического состава. [4] [10] Толщина сердцевины также значительно зависит от температуры, в частности, вблизи фазового перехода. [11]

Асимметрия [ править ]

Во многих встречающихся в природе бислоев состав створок внутренней и внешней мембраны различен. В человеческих эритроцитах внутренний (цитоплазматический) листок состоит в основном из фосфатидилэтаноламина , фосфатидилсерина и фосфатидилинозитола и его фосфорилированных производных. Напротив, внешний (внеклеточный) листок основан на фосфатидилхолине , сфингомиелине и различных гликолипидах. [12] [13] В некоторых случаях эта асимметрия основана на том, где липиды образуются в клетке, и отражает их первоначальную ориентацию. [14]Биологические функции липидной асимметрии изучены недостаточно, хотя ясно, что она используется в нескольких различных ситуациях. Например, когда клетка подвергается апоптозу , фосфатидилсерин - обычно локализованный в цитоплазматической створке - переносится на внешнюю поверхность: там он распознается макрофагом, который затем активно очищает умирающую клетку.

Асимметрия липидов возникает, по крайней мере частично, из-за того, что большинство фосфолипидов синтезируются и изначально вставляются во внутренний монослой: те, которые составляют внешний монослой, затем переносятся из внутреннего монослоя с помощью класса ферментов, называемых флиппазами . [15] [16] Другие липиды, такие как сфингомиелин, по-видимому, синтезируются во внешней створке. Флиппазы являются членами более крупного семейства молекул транспорта липидов, которое также включает флоппазы, которые переносят липиды в противоположном направлении, и скрамблазы, которые рандомизируют распределение липидов по липидным бислоям (как в апоптотических клетках). В любом случае, как только липидная асимметрия установлена, она обычно не рассеивается быстро, потому что спонтанное переключение липидов между листочками происходит очень медленно.[17]

Эту асимметрию можно имитировать в лабораторных условиях в модельных двухслойных системах. Некоторые типы очень маленьких искусственных пузырьков автоматически становятся слегка асимметричными, хотя механизм, с помощью которого возникает эта асимметрия, очень отличается от такового в клетках. [18] Используя два разных монослоя в осаждении Ленгмюра-Блоджетт [19] или комбинацию отложений Ленгмюра-Блоджетт и разрыва пузырьков [20], также можно синтезировать асимметричный плоский бислой. Эта асимметрия может быть потеряна со временем, так как липиды в поддерживаемых бислоях могут быть склонны к колебаниям. [21]

Фазы и фазовые переходы [ править ]

Диаграмма, показывающая влияние ненасыщенных липидов на бислой. Липиды с ненасыщенным хвостом (синий) нарушают упаковку липидов с только насыщенным хвостом (черный). Получающийся в результате бислой имеет больше свободного пространства и, как следствие, более проницаем для воды и других небольших молекул.
Дополнительная информация: фазовое поведение липидного бислоя

При данной температуре липидный бислой может существовать либо в жидкой, либо в гелевой (твердой) фазе. Все липиды имеют характерную температуру, при которой они переходят (плавятся) из гелевой фазы в жидкую. В обеих фазах молекулы липидов не могут перемещаться через бислой, но в жидких бислоях данный липид будет обмениваться местами со своим соседом миллионы раз в секунду. Этот случайный обмен позволяет липидам диффундировать и, таким образом, блуждать по поверхности мембраны. [22] В отличие от жидкофазных бислоев, липиды в двухслойной гелевой фазе обладают меньшей подвижностью.

Фазовое поведение липидных бислоев в значительной степени определяется силой притягивающих ван-дер-ваальсовых взаимодействий между соседними липидными молекулами. Липиды с более длинными хвостами имеют большую площадь для взаимодействия, увеличивая силу этого взаимодействия и, как следствие, уменьшая подвижность липидов. Таким образом, при данной температуре липид с коротким хвостом будет более жидким, чем идентичный липид с длинным хвостом. [10] На температуру перехода также может влиять степень ненасыщенности липидных хвостов. Ненасыщенная двойная связь может вызвать перегиб в алкане.цепи, нарушая упаковку липидов. Это нарушение создает дополнительное свободное пространство внутри бислоя, что обеспечивает дополнительную гибкость соседним цепям. [10] Пример этого эффекта можно отметить в повседневной жизни, когда сливочное масло, которое имеет большой процент насыщенных жиров, является твердым при комнатной температуре, в то время как растительное масло, которое в основном ненасыщенное, является жидким.

Большинство природных мембран представляют собой сложную смесь различных молекул липидов. Если некоторые из компонентов являются жидкими при заданной температуре, а другие находятся в гелевой фазе, эти две фазы могут сосуществовать в пространственно разделенных областях, как айсберг, плавающий в океане. Это разделение фаз играет решающую роль в биохимических явлениях, поскольку компоненты мембраны, такие как белки, могут разделяться на ту или иную фазу [23] и, таким образом, локально концентрироваться или активироваться. Одним из особенно важных компонентов многих смешанных фазовых систем является холестерин , который модулирует проницаемость двух слоев, механическую прочность и биохимические взаимодействия.

Химия поверхности [ править ]

В то время как липидные хвосты в первую очередь модулируют поведение двухслойной фазы, это головная группа, которая определяет химию поверхности бислоя. Большинство природных бислоев состоят в основном из фосфолипидов , но сфинголипиды и стерины, такие как холестерин , также являются важными компонентами. [24] Из фосфолипидов наиболее распространенной головной группой является фосфатидилхолин (PC), на долю которого приходится около половины фосфолипидов в большинстве клеток млекопитающих. [25] ПК представляет собой цвиттерионную головную группу , поскольку он имеет отрицательный заряд на фосфатной группе и положительный заряд на амине, но, поскольку эти локальные заряды уравновешивают, нет чистого заряда.

Другие головные группы также присутствуют в различной степени и могут включать фосфатидилсерин (PS), фосфатидилэтаноламин (PE) и фосфатидилглицерин (PG). Эти альтернативные головные группы часто предоставляют определенные биологические функции, которые сильно зависят от контекста. Так , например, присутствие PS на внеклеточную мембрану лице эритроцитов является маркером клеточного апоптоза , [26] , тогда как в PS рост пластины везикул необходимо для нуклеации из гидроксиапатита кристаллов и последующей минерализации костей. [27] [28]В отличие от ПК, некоторые другие головные группы несут чистый заряд, который может изменять электростатические взаимодействия малых молекул с бислоем. [29]

Биологические роли [ править ]

Сдерживание и разделение [ править ]

Основная роль липидного бислоя в биологии состоит в том, чтобы отделить водные компартменты от их окружения. Без какой-либо формы барьера, отделяющего «я» от «не-я», трудно даже определить понятие организма или жизни. Этот барьер принимает форму липидного бислоя у всех известных форм жизни, за исключением нескольких видов архей, которые используют специально адаптированный липидный монослой. [7] Было даже высказано предположение, что самой первой формой жизни могла быть простая липидная везикула с практически единственной биосинтетической способностью производить больше фосфолипидов . [30] The partitioning ability of the lipid bilayer is based on the fact that hydrophilic molecules cannot easily cross the hydrophobic bilayer core, as discussed in Transport across the bilayer below. The nucleus, mitochondria and chloroplasts have two lipid bilayers, while other sub-cellular structures are surrounded by a single lipid bilayer (such as the plasma membrane, endoplasmic reticula, Golgi apparatus and lysosomes). See Organelle.[31]

Prokaryotes have only one lipid bilayer - the cell membrane (also known as the plasma membrane). Many prokaryotes also have a cell wall, but the cell wall is composed of proteins or long chain carbohydrates, not lipids. In contrast, eukaryotes have a range of organelles including the nucleus, mitochondria, lysosomes and endoplasmic reticulum. All of these sub-cellular compartments are surrounded by one or more lipid bilayers and, together, typically comprise the majority of the bilayer area present in the cell. In liver hepatocytes for example, the plasma membrane accounts for only two percent of the total bilayer area of the cell, whereas the endoplasmic reticulum contains more than fifty percent and the mitochondria a further thirty percent.[32]

Illustration of a GPCR signaling protein. In response to a molecule such as a hormone binding to the exterior domain (blue) the GPCR changes shape and catalyzes a chemical reaction on the interior domain (red). The gray feature is the surrounding bilayer.

Signaling[edit]

See also: Neurotransmission and Lipid raft

Probably the most familiar form of cellular signaling is synaptic transmission, whereby a nerve impulse that has reached the end of one neuron is conveyed to an adjacent neuron via the release of neurotransmitters. This transmission is made possible by the action of synaptic vesicles loaded with the neurotransmitters to be released. These vesicles fuse with the cell membrane at the pre-synaptic terminal and release its contents to the exterior of the cell. The contents then diffuse across the synapse to the post-synaptic terminal.

Lipid bilayers are also involved in signal transduction through their role as the home of integral membrane proteins. This is an extremely broad and important class of biomolecule. It is estimated that up to a third of the human proteome are membrane proteins.[33] Some of these proteins are linked to the exterior of the cell membrane. An example of this is the CD59 protein, which identifies cells as “self” and thus inhibits their destruction by the immune system. The HIV virus evades the immune system in part by grafting these proteins from the host membrane onto its own surface.[32] Alternatively, some membrane proteins penetrate all the way through the bilayer and serve to relay individual signal events from the outside to the inside of the cell. The most common class of this type of protein is the G protein-coupled receptor (GPCR). GPCRs are responsible for much of the cell's ability to sense its surroundings and, because of this important role, approximately 40% of all modern drugs are targeted at GPCRs.[34]

In addition to protein- and solution-mediated processes, it is also possible for lipid bilayers to participate directly in signaling. A classic example of this is phosphatidylserine-triggered phagocytosis. Normally, phosphatidylserine is asymmetrically distributed in the cell membrane and is present only on the interior side. During programmed cell death a protein called a scramblase equilibrates this distribution, displaying phosphatidylserine on the extracellular bilayer face. The presence of phosphatidylserine then triggers phagocytosis to remove the dead or dying cell.

Characterization methods[edit]

Further information: Lipid bilayer characterization
Transmission Electron Microscope (TEM) image of a lipid vesicle. The two dark bands around the edge are the two leaflets of the bilayer. Historically, similar images confirmed that the cell membrane is a bilayer

The lipid bilayer is a very difficult structure to study because it is so thin and fragile. In spite of these limitations dozens of techniques have been developed over the last seventy years to allow investigations of its structure and function.

Electrical measurements[edit]

Electrical measurements are a straightforward way to characterize an important function of a bilayer: its ability to segregate and prevent the flow of ions in solution. By applying a voltage across the bilayer and measuring the resulting current, the resistance of the bilayer is determined. This resistance is typically quite high (108 Ohm-cm2 or more) [35] since the hydrophobic core is impermeable to charged species. The presence of even a few nanometer-scale holes results in a dramatic increase in current.[36] The sensitivity of this system is such that even the activity of single ion channels can be resolved.[37]

Fluorescence microscopy[edit]

Human red blood cells viewed through a fluorescence microscope. The cell membrane has been stained with a fluorescent dye. Scale bar is 20μm.

Electrical measurements do not provide an actual picture like imaging with a microscope can. Lipid bilayers cannot be seen in a traditional microscope because they are too thin. In order to see bilayers, researchers often use fluorescence microscopy. A sample is excited with one wavelength of light and observed in a different wavelength, so that only fluorescent molecules with a matching excitation and emission profile will be seen. Natural lipid bilayers are not fluorescent, so a dye is used that attaches to the desired molecules in the bilayer. Resolution is usually limited to a few hundred nanometers, much smaller than a typical cell but much larger than the thickness of a lipid bilayer.

Electron microscopy[edit]

Electron microscopy offers a higher resolution image. In an electron microscope, a beam of focused electrons interacts with the sample rather than a beam of light as in traditional microscopy. In conjunction with rapid freezing techniques, electron microscopy has also been used to study the mechanisms of inter- and intracellular transport, for instance in demonstrating that exocytotic vesicles are the means of chemical release at synapses.[38]

Nuclear magnetic resonance spectroscopy[edit]

31P-NMR(nuclear magnetic resonance) spectroscopy is widely used for studies of phospholipid bilayers and biological membranes in native conditions. The analysis[39] of 31P-NMR spectra of lipids could provide a wide range of information about lipid bilayer packing, phase transitions (gel phase, physiological liquid crystal phase, ripple phases, non bilayer phases), lipid head group orientation/dynamics, and elastic properties of pure lipid bilayer and as a result of binding of proteins and other biomolecules.

Atomic force microscopy[edit]

3d-Adapted AFM images showing formation of transmembrane pores (holes) in supported lipid bilayer[40]
Illustration of a typical AFM scan of a supported lipid bilayer. The pits are defects in the bilayer, exposing the smooth surface of the substrate underneath.

A new method to study lipid bilayers is Atomic force microscopy (AFM). Rather than using a beam of light or particles, a very small sharpened tip scans the surface by making physical contact with the bilayer and moving across it, like a record player needle. AFM is a promising technique because it has the potential to image with nanometer resolution at room temperature and even under water or physiological buffer, conditions necessary for natural bilayer behavior. Utilizing this capability, AFM has been used to examine dynamic bilayer behavior including the formation of transmembrane pores (holes)[40] and phase transitions in supported bilayers.[41] Another advantage is that AFM does not require fluorescent or isotopic labeling of the lipids, since the probe tip interacts mechanically with the bilayer surface. Because of this, the same scan can image both lipids and associated proteins, sometimes even with single-molecule resolution.[40][42] AFM can also probe the mechanical nature of lipid bilayers.[43]

Dual polarisation interferometry[edit]

Lipid bilayers exhibit high levels of birefringence where the refractive index in the plane of the bilayer differs from that perpendicular by as much as 0.1 refractive index units. This has been used to characterise the degree of order and disruption in bilayers using dual polarisation interferometry to understand mechanisms of protein interaction.

Quantum chemical calculations[edit]

Lipid bilayers are complicated molecular systems with many degrees of freedom. Thus, atomistic simulation of membrane and in particular ab initio calculations of its properties is difficult and computationally expensive. Quantum chemical calculations has recently been successfully performed to estimate dipole and quadrupole moments of lipid membranes.[44]

Transport across the bilayer[edit]

Passive diffusion[edit]

Most polar molecules have low solubility in the hydrocarbon core of a lipid bilayer and, as a consequence, have low permeability coefficients across the bilayer. This effect is particularly pronounced for charged species, which have even lower permeability coefficients than neutral polar molecules.[45] Anions typically have a higher rate of diffusion through bilayers than cations.[46][47] Compared to ions, water molecules actually have a relatively large permeability through the bilayer, as evidenced by osmotic swelling. When a cell or vesicle with a high interior salt concentration is placed in a solution with a low salt concentration it will swell and eventually burst. Such a result would not be observed unless water was able to pass through the bilayer with relative ease. The anomalously large permeability of water through bilayers is still not completely understood and continues to be the subject of active debate.[48] Small uncharged apolar molecules diffuse through lipid bilayers many orders of magnitude faster than ions or water. This applies both to fats and organic solvents like chloroform and ether. Regardless of their polar character larger molecules diffuse more slowly across lipid bilayers than small molecules.[49]

Structure of a potassium ion channel. The alpha helices penetrate the bilayer (boundaries indicated by red and blue lines), opening a hole through which potassium ions can flow

Ion pumps and channels[edit]

Two special classes of protein deal with the ionic gradients found across cellular and sub-cellular membranes in nature- ion channels and ion pumps. Both pumps and channels are integral membrane proteins that pass through the bilayer, but their roles are quite different. Ion pumps are the proteins that build and maintain the chemical gradients by utilizing an external energy source to move ions against the concentration gradient to an area of higher chemical potential. The energy source can be ATP, as is the case for the Na+-K+ ATPase. Alternatively, the energy source can be another chemical gradient already in place, as in the Ca2+/Na+ antiporter. It is through the action of ion pumps that cells are able to regulate pH via the pumping of protons.

In contrast to ion pumps, ion channels do not build chemical gradients but rather dissipate them in order to perform work or send a signal. Probably the most familiar and best studied example is the voltage-gated Na+ channel, which allows conduction of an action potential along neurons. All ion pumps have some sort of trigger or “gating” mechanism. In the previous example it was electrical bias, but other channels can be activated by binding a molecular agonist or through a conformational change in another nearby protein.[50]

Schematic illustration of pinocytosis, a type of endocytosis

Endocytosis and exocytosis[edit]

See also: Endocytosis and Exocytosis

Some molecules or particles are too large or too hydrophilic to pass through a lipid bilayer. Other molecules could pass through the bilayer but must be transported rapidly in such large numbers that channel-type transport is impractical. In both cases, these types of cargo can be moved across the cell membrane through fusion or budding of vesicles. When a vesicle is produced inside the cell and fuses with the plasma membrane to release its contents into the extracellular space, this process is known as exocytosis. In the reverse process, a region of the cell membrane will dimple inwards and eventually pinch off, enclosing a portion of the extracellular fluid to transport it into the cell. Endocytosis and exocytosis rely on very different molecular machinery to function, but the two processes are intimately linked and could not work without each other. The primary mechanism of this interdependence is the large amount of lipid material involved.[51] In a typical cell, an area of bilayer equivalent to the entire plasma membrane will travel through the endocytosis/exocytosis cycle in about half an hour.[52] If these two processes were not balancing each other, the cell would either balloon outward to an unmanageable size or completely deplete its plasma membrane within a short time.

Exocytosis of outer membrane vesicles (MV) liberated from inflated periplasmic pockets (p) on surface of human Salmonella 3,10:r:- pathogens docking on plasma membrane of macrophage cells (M) in chicken ileum, for host-pathogen signaling in vivo.

Exocytosis in prokaryotes: Membrane vesicular exocytosis, popularly known as membrane vesicle trafficking, a Nobel prize-winning (year, 2013) process, is traditionally regarded as a prerogative of eukaryotic cells.[53] This myth was however broken with the revelation that nanovesicles, popularly known as bacterial outer membrane vesicles, released by gram-negative microbes, translocate bacterial signal molecules to host or target cells[54] to carry out multiple processes in favour of the secreting microbe e.g., in host cell invasion[55] and microbe-environment interactions, in general.[56]

Electroporation[edit]

Further information: Electroporation

Electroporation is the rapid increase in bilayer permeability induced by the application of a large artificial electric field across the membrane. Experimentally, electroporation is used to introduce hydrophilic molecules into cells. It is a particularly useful technique for large highly charged molecules such as DNA, which would never passively diffuse across the hydrophobic bilayer core.[57] Because of this, electroporation is one of the key methods of transfection as well as bacterial transformation. It has even been proposed that electroporation resulting from lightning strikes could be a mechanism of natural horizontal gene transfer.[58]

This increase in permeability primarily affects transport of ions and other hydrated species, indicating that the mechanism is the creation of nm-scale water-filled holes in the membrane. Although electroporation and dielectric breakdown both result from application of an electric field, the mechanisms involved are fundamentally different. In dielectric breakdown the barrier material is ionized, creating a conductive pathway. The material alteration is thus chemical in nature. In contrast, during electroporation the lipid molecules are not chemically altered but simply shift position, opening up a pore that acts as the conductive pathway through the bilayer as it is filled with water.

Mechanics[edit]

Further information: Lipid bilayer mechanics
Schematic showing two possible conformations of the lipids at the edge of a pore. In the top image the lipids have not rearranged, so the pore wall is hydrophobic. In the bottom image some of the lipid heads have bent over, so the pore wall is hydrophilic.

Lipid bilayers are large enough structures to have some of the mechanical properties of liquids or solids. The area compression modulus Ka, bending modulus Kb, and edge energy , can be used to describe them. Solid lipid bilayers also have a shear modulus, but like any liquid, the shear modulus is zero for fluid bilayers. These mechanical properties affect how the membrane functions. Ka and Kb affect the ability of proteins and small molecules to insert into the bilayer,[59][60] and bilayer mechanical properties have been shown to alter the function of mechanically activated ion channels.[61] Bilayer mechanical properties also govern what types of stress a cell can withstand without tearing. Although lipid bilayers can easily bend, most cannot stretch more than a few percent before rupturing.[62]

As discussed in the Structure and organization section, the hydrophobic attraction of lipid tails in water is the primary force holding lipid bilayers together. Thus, the elastic modulus of the bilayer is primarily determined by how much extra area is exposed to water when the lipid molecules are stretched apart.[63] It is not surprising given this understanding of the forces involved that studies have shown that Ka varies strongly with osmotic pressure[64] but only weakly with tail length and unsaturation.[10] Because the forces involved are so small, it is difficult to experimentally determine Ka. Most techniques require sophisticated microscopy and very sensitive measurement equipment.[43][65]

In contrast to Ka, which is a measure of how much energy is needed to stretch the bilayer, Kb is a measure of how much energy is needed to bend or flex the bilayer. Formally, bending modulus is defined as the energy required to deform a membrane from its intrinsic curvature to some other curvature. Intrinsic curvature is defined by the ratio of the diameter of the head group to that of the tail group. For two-tailed PC lipids, this ratio is nearly one so the intrinsic curvature is nearly zero. If a particular lipid has too large a deviation from zero intrinsic curvature it will not form a bilayer and will instead form other phases such as micelles or inverted micelles. Addition of small hydrophilic molecules like sucrose into mixed lipid lamellar liposomes made from galactolipid-rich thylakoid membranes destabilises bilayers into micellar phase.[66] Typically, Kb is not measured experimentally but rather is calculated from measurements of Ka and bilayer thickness, since the three parameters are related.

is a measure of how much energy it takes to expose a bilayer edge to water by tearing the bilayer or creating a hole in it. The origin of this energy is the fact that creating such an interface exposes some of the lipid tails to water, but the exact orientation of these border lipids is unknown. There is some evidence that both hydrophobic (tails straight) and hydrophilic (heads curved around) pores can coexist.[67][68]

Fusion[edit]

See also: Lipid bilayer fusion and Interbilayer forces in membrane fusion
Illustration of lipid vesicles fusing showing two possible outcomes: hemifusion and full fusion. In hemifusion, only the outer bilayer leaflets mix. In full fusion both leaflets as well as the internal contents mix.

Fusion is the process by which two lipid bilayers merge, resulting in one connected structure. If this fusion proceeds completely through both leaflets of both bilayers, a water-filled bridge is formed and the solutions contained by the bilayers can mix. Alternatively, if only one leaflet from each bilayer is involved in the fusion process, the bilayers are said to be hemifused. Fusion is involved in many cellular processes, in particular in eukaryotes, since the eukaryotic cell is extensively sub-divided by lipid bilayer membranes. Exocytosis, fertilization of an egg by sperm activation, and transport of waste products to the lysozome are a few of the many eukaryotic processes that rely on some form of fusion. Even the entry of pathogens can be governed by fusion, as many bilayer-coated viruses have dedicated fusion proteins to gain entry into the host cell.

There are four fundamental steps in the fusion process.[25] First, the involved membranes must aggregate, approaching each other to within several nanometers. Second, the two bilayers must come into very close contact (within a few angstroms). To achieve this close contact, the two surfaces must become at least partially dehydrated, as the bound surface water normally present causes bilayers to strongly repel. The presence of ions, in particular divalent cations like magnesium and calcium, strongly affects this step.[69][70] One of the critical roles of calcium in the body is regulating membrane fusion. Third, a destabilization must form at one point between the two bilayers, locally distorting their structures. The exact nature of this distortion is not known. One theory is that a highly curved "stalk" must form between the two bilayers.[71] Proponents of this theory believe that it explains why phosphatidylethanolamine, a highly curved lipid, promotes fusion.[72] Finally, in the last step of fusion, this point defect grows and the components of the two bilayers mix and diffuse away from the site of contact.

Schematic illustration of the process of fusion through stalk formation.
Diagram of the action of SNARE proteins docking a vesicle for exocytosis. Complementary versions of the protein on the vesicle and the target membrane bind and wrap around each other, drawing the two bilayers close together in the process.[73]

The situation is further complicated when considering fusion in vivo since biological fusion is almost always regulated by the action of membrane-associated proteins. The first of these proteins to be studied were the viral fusion proteins, which allow an enveloped virus to insert its genetic material into the host cell (enveloped viruses are those surrounded by a lipid bilayer; some others have only a protein coat). Eukaryotic cells also use fusion proteins, the best-studied of which are the SNAREs. SNARE proteins are used to direct all vesicular intracellular trafficking. Despite years of study, much is still unknown about the function of this protein class. In fact, there is still an active debate regarding whether SNAREs are linked to early docking or participate later in the fusion process by facilitating hemifusion.[74]

In studies of molecular and cellular biology it is often desirable to artificially induce fusion. The addition of polyethylene glycol (PEG) causes fusion without significant aggregation or biochemical disruption. This procedure is now used extensively, for example by fusing B-cells with myeloma cells.[75] The resulting “hybridoma” from this combination expresses a desired antibody as determined by the B-cell involved, but is immortalized due to the melanoma component. Fusion can also be artificially induced through electroporation in a process known as electrofusion. It is believed that this phenomenon results from the energetically active edges formed during electroporation, which can act as the local defect point to nucleate stalk growth between two bilayers.[76]

Model systems[edit]

Further information: Model lipid bilayers

Lipid bilayers can be created artificially in the lab to allow researchers to perform experiments that cannot be done with natural bilayers. They can also be used in the field of Synthetic Biology, to define the boundaries of artificial cells. These synthetic systems are called model lipid bilayers. There are many different types of model bilayers, each having experimental advantages and disadvantages. They can be made with either synthetic or natural lipids. Among the most common model systems are:

  • Black lipid membranes (BLM)
  • Supported lipid bilayers (SLB)
  • Tethered Bilayer Lipid Membranes (t-BLM)
  • Vesicles
  • Droplet Interface Bilayers (DIBs)

Commercial applications[edit]

To date, the most successful commercial application of lipid bilayers has been the use of liposomes for drug delivery, especially for cancer treatment. (Note- the term “liposome” is in essence synonymous with “vesicle” except that vesicle is a general term for the structure whereas liposome refers to only artificial not natural vesicles) The basic idea of liposomal drug delivery is that the drug is encapsulated in solution inside the liposome then injected into the patient. These drug-loaded liposomes travel through the system until they bind at the target site and rupture, releasing the drug. In theory, liposomes should make an ideal drug delivery system since they can isolate nearly any hydrophilic drug, can be grafted with molecules to target specific tissues and can be relatively non-toxic since the body possesses biochemical pathways for degrading lipids.[77]

The first generation of drug delivery liposomes had a simple lipid composition and suffered from several limitations. Circulation in the bloodstream was extremely limited due to both renal clearing and phagocytosis. Refinement of the lipid composition to tune fluidity, surface charge density, and surface hydration resulted in vesicles that adsorb fewer proteins from serum and thus are less readily recognized by the immune system.[78] The most significant advance in this area was the grafting of polyethylene glycol (PEG) onto the liposome surface to produce “stealth” vesicles, which circulate over long times without immune or renal clearing.[79]

The first stealth liposomes were passively targeted at tumor tissues. Because tumors induce rapid and uncontrolled angiogenesis they are especially “leaky” and allow liposomes to exit the bloodstream at a much higher rate than normal tissue would.[80] More recently[when?] work has been undertaken to graft antibodies or other molecular markers onto the liposome surface in the hope of actively binding them to a specific cell or tissue type.[81] Some examples of this approach are already in clinical trials.[82]

Another potential application of lipid bilayers is the field of biosensors. Since the lipid bilayer is the barrier between the interior and exterior of the cell, it is also the site of extensive signal transduction. Researchers over the years have tried to harness this potential to develop a bilayer-based device for clinical diagnosis or bioterrorism detection. Progress has been slow in this area and, although a few companies have developed automated lipid-based detection systems, they are still targeted at the research community. These include Biacore (now GE Healthcare Life Sciences), which offers a disposable chip for utilizing lipid bilayers in studies of binding kinetics[83] and Nanion Inc., which has developed an automated patch clamping system.[84] Other, more exotic applications are also being pursued such as the use of lipid bilayer membrane pores for DNA sequencing by Oxford Nanolabs. To date, this technology has not proven commercially viable.

A supported lipid bilayer (SLB) as described above has achieved commercial success as a screening technique to measure the permeability of drugs. This parallel artificial membrane permeability assay PAMPA technique measures the permeability across specifically formulated lipid cocktail(s) found to be highly correlated with Caco-2 cultures,[85][86] the gastrointestinal tract,[87] blood–brain barrier[88] and skin.[89]

History[edit]

Further information: History of cell membrane theory

By the early twentieth century scientists had come to believe that cells are surrounded by a thin oil-like barrier,[90] but the structural nature of this membrane was not known. Two experiments in 1925 laid the groundwork to fill in this gap. By measuring the capacitance of erythrocyte solutions, Hugo Fricke determined that the cell membrane was 3.3 nm thick.[91]

Although the results of this experiment were accurate, Fricke misinterpreted the data to mean that the cell membrane is a single molecular layer. Prof. Dr. Evert Gorter[92] (1881–1954) and F. Grendel of Leiden University approached the problem from a different perspective, spreading the erythrocyte lipids as a monolayer on a Langmuir-Blodgett trough. When they compared the area of the monolayer to the surface area of the cells, they found a ratio of two to one.[93] Later analyses showed several errors and incorrect assumptions with this experiment but, serendipitously, these errors canceled out and from this flawed data Gorter and Grendel drew the correct conclusion- that the cell membrane is a lipid bilayer.[25]

This theory was confirmed through the use of electron microscopy in the late 1950s. Although he did not publish the first electron microscopy study of lipid bilayers[94] J. David Robertson was the first to assert that the two dark electron-dense bands were the headgroups and associated proteins of two apposed lipid monolayers.[95][96] In this body of work, Robertson put forward the concept of the “unit membrane.” This was the first time the bilayer structure had been universally assigned to all cell membranes as well as organelle membranes.

Around the same time, the development of model membranes confirmed that the lipid bilayer is a stable structure that can exist independent of proteins. By “painting” a solution of lipid in organic solvent across an aperture, Mueller and Rudin were able to create an artificial bilayer and determine that this exhibited lateral fluidity, high electrical resistance and self-healing in response to puncture,[97] all of which are properties of a natural cell membrane. A few years later, Alec Bangham showed that bilayers, in the form of lipid vesicles, could also be formed simply by exposing a dried lipid sample to water.[98] This was an important advance, since it demonstrated that lipid bilayers form spontaneously via self assembly and do not require a patterned support structure.

In 1977, a totally synthetic bilayer membrane was prepared by Kunitake and Okahata, from a single organic compound, didodecyldimethylammonium bromide.[99] It clearly shows that the bilayer membrane was assembled by the van der Waals interaction.

See also[edit]

  • Surfactant
  • Membrane biophysics
  • Lipid polymorphism
  • Lipidomics

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External links[edit]

  • Avanti Lipids One of the largest commercial suppliers of lipids. Technical information on lipid properties and handling and lipid bilayer preparation techniques.
  • LIPIDAT An extensive database of lipid physical properties
  • Structure of Fluid Lipid Bilayers Simulations and publication links related to the cross sectional structure of lipid bilayers.
  • Lipid Bilayers and the Gramicidin Channel (requires Java plugin) Pictures and movies showing the results of molecular dynamics simulations of lipid bilayers.
  • Structure of Fluid Lipid Bilayers, from the Stephen White laboratory at University of California, Irvine
  • Animations of lipid bilayer dynamics (requires Flash plugin)