cell biology 6 Flashcards
Epithelial cells
There are many different epithelial tissues in the body, including the G.I. tract, kidneys, all exocrine glands, gall bladder, choroid plexus, ciliary body, corneal epithelium, and mucous membranes (but not the lungs ). Epithelial cells are organized into sheets of cells, often forming tubular structures, like the epithelium lining the G.I. tract. The apical surface faces the ‘special’ fluid (e.g., food in the gut, urine in the kidney, saliva in the parotid duct) and usually contains the special transporters that endow the epithelium with its specialized transport properties. The basolateral surface is exposed to the interstitial fluid and usually has generic transport properties like the plasma membranes of non-epithelial cells (neurons, for example). Synonyms for apical include mucosal and lumenal; synonyms for basolateral include serosal and peritubular. The cells that compose the epithelial sheet are glued to their neighbors by tight junctions. The glue is indeed tight is some epithelia, such as sweat glands and the distal parts of kidney tubules, preventing virtually any substance from passing from one side to the other by passing in between the cells. In most epithelia, however, the tight junctions are not very tight. For example, tight junctions of the small and large intestine, gall bladder, and proximal part of the kidney tubules are relatively leaky, and provide a pericellular shunt pathway for the movement of water and solutes. Leaky tight junctions are somewhat selective in their leakiness; some are relatively more permeable to cations, others to anions.
transport in the lungs
The main task of the lungs is to transport O2 into and CO2 out of the blood. There are no membrane transporters (active or otherwise) for O2 and CO2; they are lipid soluble molecules that diffuse freely through all membranes. Thus, the endothelial cells in the lung that separate blood from air in the alveoli of the lungs do not possess special transporters like those in epithelia to transport O2 or CO2.
Epithelial Transport
In general, epithelia engaged in massive transport of substances are leaky, while those epithelia doing the finishing work (‘fine tuning’) are tight. Leaky epithelia cannot maintain energy gradients as large as those produced by tight epithelia, because solutes and water leak back across the epithelium through the pericellular shunt. Thus, to get across an epithelium, a substance must follow one (or both) of two possible routes: it may either cross two membranes by entering the epithelial cell on one side and leaving on the other, or it may cross no membranes at all by passing in between cells through the pericellular shunt pathway. The driving force for nearly all transport – water, salts, nutrients, non-volatile metabolic wastes – is the Na/K pump (always located in the basolateral membrane). By keeping intracellular sodium ion concentration low, the Na/K pump provides the energy to drive a host of secondary transporters. Protons are the main exception to the otherwise universal dependence of epithelial pumping on the Na/K pump, because primary active transporters have evolved for protons, most notably in the stomach (to secrete acid into the lumen of the stomach) and kidney (to excrete protons, which are a metabolic waste product.)
basolateral membrane
surface is exposed to the interstitial fluid and usually has generic transport properties like the plasma membranes of non-epithelial cells Note that the basolateral membrane is just like many other cells, containing relatively low sodium permeability and high potassium permeability. What value of membrane potential would you expect to record across the basolateral membrane? Answer: because it’s like the plasma membrane of a neuron, Vm would be about -70 mV. The basolateral membrane also contains some chloride channels, and the Na/K pump.
apical membrane
faces the ‘special’ fluid (e.g., food in the gut, urine in the kidney, saliva in the parotid duct) and usually contains the special transporters that endow the epithelium with its specialized transport properties. It is relatively highly permeable to sodium, not potassium. What would be the potential across this membrane? (Answer: Vm would be more positive, perhaps +10 mV.) In addition, there is no Na/K pump in the apical membrane.
how salt and water are transported from apical to basolateral solution
Assume that identical NaCl solutions are placed on either side of the epithelium. Sodium ions leak into the cell across the apical membrane, down their electrochemical gradient. They are then pumped out of the other side of the cell by the Na/K pump, across the basolateral membrane. This results in the net transport across the epithelium of a positive charge, and chloride follows passively, drawn by the electrical force. The net transport of NaCl produces an osmotic gradient, which in turn draws water along. What would happen to the transport if the Na/K pump were blocked? As the cell filled with sodium (leaking in across the apical membrane), the driving force for further sodium entry across the apical membrane would be reduced, and net transport of sodium, chloride, and water would decrease.
What is the transepithelial potential difference (transPD)?
TransPD = Vm (Basolateral) – Vm (Apical). Keep in mind a couple of rules: i) all membrane potentials are written as the potential of the inside of the cell with respect to the outside (i.e., outside = zero); ii) the transepithelial potential is written as the potential of the apical solution with respect to the basolateral (i.e., basolateral = zero); iii) the cell is isopotential (all voltage drops are at membranes, so all lines showing electric potential are horizontal – no change over distance, except across membranes). Start at the basolateral side. The basolateral solution is defined as zero. You are given that Vm (B) = -50 mV, so in crossing the basolateral membrane from outside to inside, you must drop down 50 mV. The cell is isopotential (this is only an approximation, but a good one), so the line across the cell is horizontal. In leaving the cell by crossing the apical membrane, you must change by 10 mV, because Vm (A) = +10 mV. Do you move up or down? Because the inside is 10 mV more positive than the outside, you must move down, emerging at a potential of - 60 mV, compared to the basolateral solution.
leaky epithelium
allows significant fluxes through the paracellular shunt pathway. This would be characteristic of an epithelium like the one lining the G.I. tract, where relatively large fluxes occur. The shunt pathway in this case is relatively more permeable to chloride than to sodium, but is by no means totally impermeable to sodium (the molecular mechanisms by which tight junctions achieve selective permeabilities are not well understood). The major difference is that the leaky epithelium is usually capable of moving a larger amount of material. In addition, the leaky tight junctions partially ‘short out’ the transepithelial potential difference, so that a lower transepithelial voltage would be measured.
electroneutral cotransporter
e.g. carries one potassium, two chloride, and one sodium ions into the cell each cycle (it sounds more like pinocytosis than membrane transport!). This transporter is found in several important epithelia, including the kidney loop of Henle and the respiratory tract. Qualitatively, though, the end result is no different than other scenerios.
Secretion of fluid by epithelia
Not all cells in a given epithelium are identical. On the contrary, individual cells may be highly specialized, some for absorption, some for secretion. The mechanisms described so far concern absorption by epithelia – moving solutes from lumen to blood. Things can travel the other direction, too. One secretion pathway is of special clinical importance, for it underlies important diseases, including some types of diarrhea (including the worst of all, cholera), and also cystic fibrosis. The key to understanding epithelial secretion in general and the main defect in those diseases is a chloride channel in the apical membrane (especially in the GI tract and lungs). Normally, in a resting cell, this Cl- channel is closed. However, when this Cl- channel is activated, the cell begins to secrete electrolytes and water into the lumen. The secretion is driven by Cl- leaking out of the cell into the lumen. The Cl- concentration in the cell is high, thanks to a Cl- pump in the basolateral membrane. The pump is a Na-K-2Cl cotransporter that uses the downhill leakage of Na+ into the cell to drive the uptake of Cl- from the interstitial fluid. As the Cl- leaks out of the cell into the lumen, the electrical negativity that it creates draws Na+ along passively. The Na+ flows mostly through the intercellular shunt pathway. The resulting osmotic gradient draws water along, too, giving a net secretion of an isotonic solution of NaCl.
Turning on the Cl- channel
So eptithelia have mechanisms both to absorb and to secrete. Which one wins? At rest, the apical Cl- channels are closed, so absorption wins. A variety of stimuli can open the Cl- channel and turn on secretion. In the GI tract, it happens physiologically during digestion (parasympathetic nerve stimulation, hormones in the blood), as chemicals activate receptors in the basolateral membrane (the signal is carried across the inside of the cell from the basolateral receptors to the apical membrane by ‘cell signaling’ mechanisms (Ca++ ions, activated protein kinases, cyclic AMP, etc.). Pathogens also can activate the Cl- channels. Cholera toxin, for example, acts by locking open this channel, causing a massive efflux of fluid from the cell, leading to profound diarrhea and dehydration. While there are probably multiple types of Cl- channels (different gene products) involved in fluid secretion by epithelia, one of the most important is the ‘Cystic Fibrosis Transmembrane Conductance Regulator.’ (CFTCR, or more commonly CFTR). Cystic fibrosis is a genetic disease; it is this Cl- channel that is mutated, which reduces the ability of epithelia to secrete ‘serous’ (watery) fluid, leading to thickened mucous secretions, infections, and other life-shortening complications.
Absorption of nutrients
In the G.I. tract, enzymes secreted by digestive glands hydrolyze ingested proteins and polysaccharides, and the resulting amino acids and sugars are pumped from the G.I. lumen into the blood by the G.I. epithelium. Virtually identical mechanisms operate in the kidney, where glucose and amino acids are reabsorbed by the epithelial cells of the proximal tubule after being filtered from the plasma in the glomerulus. each nutrient is pumped across the apical membrane, and then passively moves out of the cell into the interstitial fluid. The sugar and amino acid pumps are examples of sodium-dependent secondary active transport systems. If [Na+] in the mucosal solution is removed (replaced by non-permeating choline+), sugar and amino acid pumping stops. Conversely, removing sugars and amino acids reduces the movement of Na+ from mucosal to serosal fluid. The transporter captures some of the energy released as Na+ moves down its electrochemical gradient into the cell, and uses this energy to pump the sugar or amino acid against its gradient into the cell. Note that the transport of glucose by epithelial apical membranes is different from the transport across the plasma membranes of nonepithelial cells (e.g., muscle), where glucose is transported by “facilitated diffusion”, which is not a pump.
regulation of absorbing nutrients
In one sense, the transporters are not regulated, at least not by the ECF composition. Instead, they seem geared for maximum transport of nutrients any time, day or night. Thus, if a person drinks a glass of water, whether thirsty or not, all of the water will be absorbed by the G.I. epithelium and put into the blood. If a person eats five grams (that’s a lot) of table salt, virtually all of it enters the blood plasma. Same for glucose, and virtually all other common nutrients. (There is great chemical specificity of the transporters, however. For example, L-amino acids and D-sugars are selectively transported, but not their stereoisomers. Eating L-glucose or D-amino acids (which are not absorbed) is likely to produce an osmotic diarrhea as the unabsorbable solutes suck water into the GI lumen.) The G.I. tract works so avidly probably because the transport mechanisms evolved in far less abundant environments than the one we live in, nutritionally speaking, and it was adaptive to be able to absorb all available nutrients. By not regulating ECF composition at the input end, it falls by necessity to the kidneys, at the output end, to regulate the composition of the ECF. Not very efficient.
Transport of Water
It is curious, given the importance of water, that no water pumps exist in the body (or anywhere else). This means that water always moves passively down osmotic gradients. As described above, absorption (by active transport) of salts, sugars, amino acids, and other solutes by the G.I. tract epithelium is accompanied by water absorption (as the solutes are absorbed, the lumenal contents become slightly hypo-osmotic to plasma, and so water is absorbed, always passively). In a few places (sweat gland ducts, some parts of tubules in the kidney), epithelia are relatively impermeable to water. In these special cases, osmotic gradients can be maintained by pumping solute across the water-impermeable epithelium. Consider, for example, sweat glands. Their job is to ‘excrete heat’. To do that, they deliver water to the body surface where its evaporation (an endothermic process) cools the body. Nascent sweat forms deep in the gland (by a secretory process); it has a salt composition that is similar to plasma (about 300 mosM, mainly NaCl). As the fluid travels along the sweat duct on its way to the surface, NaCl is (re)absorbed by the epithelium lining the duct and returned to the blood. Because the ducts are impermeable to water, water cannot follow the salt, and the fluid in the lumen of the duct becomes more and more dilute (it can be as low as about 50 mosM). This is useful, because the solute in sweat does not help at all in the process of heat loss, and just leaves an unsightly white crust on your tee shirt.
Ridding the body of metabolic wastes
Each day the cells in an adult human produce about 15 moles of metabolic waste solute. That is enough to more than double the osmolarity of the body fluids! (45 liters of body fluids * 0.3 osmoles/liter = 13.5 osmoles present in body fluids at any one time). And yet, a rise in plasma osmolarity of a few percent is enough to make a person thirsty, and severe dehydration raises plasma osmolarity by less than ten percent. In other words, there is a stupendous amount of metabolic waste to get rid of each day. The problem is largely solved by an extraordinarily simple chemical fact: the end product of carbon metabolism, CO2, which composes 14.5 of the 15 moles of waste, is volatile. Consequently, it is simply exhaled via the lungs, and requires no special transporters at all (CO2 permeates all membranes easily, by dissolving in them). A little water - less than a liter – is lost as exhaled water vapor with the CO2 . What about the remaining 0.5 moles (500 mmoles) of metabolic waste? They are non- volatile molecules, and of course cannot be expelled by the lungs. Even though they account for a small fraction of the total, getting rid of these non-volatile metabolic waste products is a big problem. It falls to the magnificent kidney to solve the problem, and while it is responsible for many other tasks (such as regulating the concentration of just about everything in the ECF), the most important function of the kidney is to get rid of these non-volatile metabolic wastes, because no other organ can do it. When kidney function is lost, death from uremia can follow. Uremia literally means “urine in the blood.”
What kinds of metabolic waste molecules are non-volatile?
The majority (about 450 of the 500 mmoles) are the end product of nitrogen metabolism, urea. Most of the remainder (about 50 mmoles) are protons, H+.
How does the kidney do it?
The fundamental anatomical arrangement of the kidney is just like the lungs, which get rid of the volatile waste product, CO2. That is, in both the lungs and the kidney, blood capillaries pass close to the ends of dead-end tubules (glomeruli in the kidney, alveoli in the lungs), and various chemical substances move from the blood into the tubules, eventually becoming urine in the kidney, and expired air in the lungs. Functionally speaking, however, matters are entirely different in the two organs. In the lungs, CO2 diffuses passively from blood to air. The equivalent arrangement in the kidney would be to have molecular transporters at the blood-tubule interface, and have them pump metabolic wastes (mostly urea) out of the blood, into the tubules, and letting them, together with some water, pass on. However, urea transporters do not exist. Consequently, rather than trying selectively to pump waste products out of the plasma, it forms an ultrafiltrate of plasma in the glomerulus, which contains water, salts, sugars, amino acids, and all other beneficial compounds, as well as the non-volatile metabolic waste products. Then, as this plasma ultrafiltrate passes along the renal tubules, the epithelial cells lining the tubules reabsorb (pump back into the blood) the things that it wants to keep (glucose, salts, bicarbonate, etc), allowing the wastes to pass on. It’s incredibly expensive, energetically speaking, to do it this way, requiring a great deal of ATP to drive the reabsorbing pumps. That’s enough responsibility for any organ, but the kidney does so much more. Earlier we noted that the kidney regulates the ECF composition (by adjusting the activity of the transporters that do the reabsorbing). This additional chore arises because the undisciplined GI tract absorbs just about everything presented to it (see box below), regardless of the needs of the ECF. So the list of functions of the kidney includes excreting non-volatile metabolic wastes and regulating the composition of virtually all ECF solutes – nutrients and electrolytes – as well as water.
the GI tract
While its output, feces, comprises mainly bacteria and substances eaten but not absorbable, about 30 mmoles per day of non-volatile metabolic wastes are excreted via the G.I. tract (compared to 500 by the kidney). These wastes are mostly breakdown products of red blood cells (delivered from the liver to the GI lumen), and are highly toxic if not promptly eliminated. Moreover, as noted earlier, the transporters in the GI tract are incredibly specific. For example, the stereo isomer D-glucose in absorbed, but L-glucose is not. On the other hand, while most big molecules (e.g., whole proteins) that we eat are of course not absorbed intact, but are broken down (digested) to much smaller molecules (e.g., amino acids) before being absorbed into the blood, the most deadly substance on earth, botulinum toxin, if ingested in food-gone-bad, is absorbed as an intact protein (and it’s a big protein), and once in the blood it goes about its fiendish business.
water balance
Naturally, the kidney regulates water (like everything else) balance in the body. Getting rid of extra water is relatively easy: solutes in the lumen of the kidney tubule are reabsorbed as usual, but the epithelium is made water-impermeable, so water cannot follow the solutes osmotically. Thus, the extra water stays in the lumen, and passes into the urine. (The epithelium is made impermeable to water when the hypothalamus, sensing extra water on board as a drop in plasma osmolarity, stops secreting anti-diuretic hormone (vasopressin), which causes certain kidney epithelial cells to remove water channels (aquaporins) from their apical membranes, thereby reducing water reabsorption.) Conserving water in times of dehydration, like when stranded in the desert, is another matter altogether. Remember, there are no water pumps, so the kidney cannot just pump water back into the blood from the tubules. This makes for a huge physiological problem. You can guess what organ has had to solve the problem. Lacking water pumps, the renal tubules have had to evolve a far more complicated mechanism to conserve water. It works pretty well: human urine can be as concentrated as 1200 mosM.
Conserving water
Conceptually, the simplest way would be to pump water out of the ultrafiltrate in the renal tubules, back into the blood. But water pumps do not exist in biology, so a more complex process has evolved that accomplishes the same thing. It involves separating in space the removal of solute and the removal of water from the ultrafiltrate in the tubule. There are three steps to the process. First, NaCl is actively transported out of the ascending limb of the loop of Henle as it rises through the renal medulla. The tubule here has a very low permeability to water, so the fluid in the tubule lumen becomes hypo-osmotic to plasma (it’s about 50 mosM at the top of the loop). In addition, the surrounding interstitial fluid is poorly vascularized in the medulla, so the salt accumulates in the ECF and is not washed away, creating a hyperosmotic interstitium. This is a key point. Second, the distal tubule is permeable to water, and the interstitium here (in the renal cortex) is well vascularized. Consequently, water passively leaves the tubule and is returned to the blood. Thus, the fluid arriving at the end of the distal tubule is isosmotic with plasma (about 300 mosM). The volume of fluid in the lumen has been greatly reduced though. In other words, five- sixths of the salt was removed by the ascending loop of Henle (remember: no volume change, because no water movement), and five-sixths of the water (and volume) left the distal tubule and returned to the blood. to the blood. Third, the tubule (now called the collecting duct) plunges back down into the renal medulla, through the hyperosmotic interstitium. The fluid in the lumen is isosmotic with plasma as it begins its passage. But the collecting duct tubule is permeable to water, and so water leaves the lumen, thereby making the lumenal fluid (urine) hyperosmotic to plasma. (Of course, water leaving the collecting duct will dilute the medullary interstitium, partially defeating the system. But because most (about 5/6) of the water was previously removed, the amount of water entering the collecting duct is greatly reduced.) In summary, the keys to this ingenious device is that most salt and water are removed from the tubule at separate locations, and the salt is kept around to provide a special, hyperosmotic interstitium to draw a little extra water from the lumen of the collecting duct. All because there are no water pumps.
what happens is permeability for Na increases?
(this happens during an action potential). We can say right away that EK and ENa will not change (no changes in concentrations, at least in the short term). To say it in words, when PNa increases, Na+ ions are able to move into the cell much more easily; the inward movement of positive charge depolarizes the membrane. As Vm moves closer to ENa, the driving force on the Na+ ions is reduced, while the driving force on K+ is increased as Vm moves away from EK (remember, the driving force on any ion is just the difference between Vm and the ion’s equilibrium potential). The new steady potential is reached when the total amount of Na+ entering equals the total amount of K+ leaving the cell. In other words, when the Na+ and K+ currents are equal and opposite, Vm doesn’t change.
what happens if external Na is reduced? (that is, most Na+ is replaced with a nonpermeating cation, such as choline+)
ENa will move closer to zero, because the change in [Na+]o has made the Na+ concentrations in the ICF and ECF more nearly equal (when [Na+]o= [Na+]i, ENa= 0, because the log of 1 is zero). From the Nernst equation, ENa= 0 mV.
what happens if external K is increased?
Intuitively, we can see that an increase in [K+]o will reduce the efflux of K+ from the cell. Because less K+ leaves, the cell will depolarize. EK will move closer to zero (we have made the internal and external concentrations more nearly equal); from the Nernst equation: EK= -60 mV. That’s a big change (almost 30 mV), and reflects the fact that the ratio of [K+]o to [K+]i determines EK, and we have tripled the ratio with only a 10 mM change in [K+]o. Clinically, this can be very important. First, it’s easy to imagine how blood potassium could increase if a little bit of the K+ leaked out of cells (remember, over 98% of the total K+ in the body is in the ICF). And second, a 20-30 mV depolarization of cells in the heart can quickly lead to cardiac arrest. In summary, external Na+ has little effect, and external K+ has a marked effect on membrane potential in nerve and muscle cells. This is simply because the plasma membrane of nerve cells and muscle cells is much more permeable to K+ than to any other ion: wherever EK goes, Vm follows in these cells.
Clinical notes on Hyperkalemia
a rather modest rise in ECF potassium ion concentration has a big effect on Vm. This can have serious clinical consequences. The reason that such a small change (only 4 mM) has such a big effect goes back to the Nernst equation: the potassium equilibrium potential, EK, depends on the ratio of external to internal potassium ion concentrations, and the ECF potassium concentration has doubled. So we expect a big change in EK. In cells that have a relatively high potassium permeability, Vm will slavishly follow EK, and so the membrane potential will depolarize a lot. A large depolarization of cells can be life threatening. In acute hyperkalemia, the main danger concerns the reliable conduction of electircal signals (action potentials) in the heart. As you will study a little later, the heart relies on a special electrical conduction system (intrinsic to the heart) to coordinate the contraction of its muscle fibers each heartbeat. These synchronized electrical signals can become disrupted during acute hyperkalemia, causing cardiac arrhythmias as conduction blocks occur and maverick pacemakers arise in various locations of the conduction system. The causes of hyperkalemia, as you might expect (knowing that 98% of all of the potassium in the body fluids is in the ICF) mostly concern loss of potassium from cells. Crush injuries, burns, and other trauma that disrupt cell membranes can do it. So can immunological attack of red blood cells (causing hemolysis). One of the most important determinants of the clinical course of the hyperkalemia is the status of the kidney, whose normal job it is to excrete excess potassium. If kidney function is compromised, hyperkalemia can be much more serious than if the kidney is functioning normally.
diagnosis of hyperkalemia
usually is via an electrocardiogram (EKG) to detect cardiac arrhythmias, followed by measuring plasma potassium ion concentration.
treatment of acute hyperkalemia
involves attempts first to relieve any cardiac arrhythmias, usually by giving intravenous calcium ions (you will study the mechanism by which calcium ions quiet the conduction system later). Next, efforts are made to reduce the concentration of potassium in the plasma. This can be done by encouraging cells to take up potassium by alkalinizing the blood by giving sodium bicarbonate (Alka Seltzer), or by juicing up the energy supply (ATP) for the sodium-potassium pump by giving insulin and glucose. Finally, potassium can be removed from the body by administering an ion exchanger (oral or enema) like Kayexalate (the exchanger is a big anion that is given as the sodium salt, but it has a higher affinity for potassium than it does for sodium, so the it selectively binds up potassium ions). A nice mnemonic always helps: when you CBIGK in a patient, what should you give, in order? (answer: Calcium, Bicarb, Insulin + Glucose, Kayexalate). In actual practice, bicarbonate is used less often. Finally, a more drastic way to cleanse the blood of potassium is dialysis by an ‘artificial kidney’. Blood is taken out of the body and passed through plastic capillary tubing, which allows free exchange of ions and small molecules between the blood and the dialysate that bathes the tubing. Because the dialysate contains a lower concentration of potassium than the plasma, potassium leaves the blood, which is then returned to the patient, cleansed artificially of its excess potassium ions. (Of course, the dialysate also contains normal concentrations of other salts.)
facilitated diffusion
Some transporters act like ion channels, shuttling a single solute species in either direction. (a historic term applied to molecules that shouldn’t be able to diffuse across lipid membranes because of their large size or charge, but do get across). The best known example of this is the glucose transporter. The glucose transporter will transport glucose in either direction, and burns no energy in the process. Thus, it is not a pump. You might then wonder how cells accumulate glucose. The answer is that as soon as a glucose molecule gets into the cell, it is phosphorylated to Glucose-6-Phosphate, which doesn’t fit on the transporter, and so is “trapped” inside. Glucose uptake by cells is regulated by insulin, a hormone secreted by specialized cells in the pancreas when plasma glucose levels rise. How does insulin turn on the transporter? It turns out that in the absence of glucose, the transporter is not even present in the plasma membrane; it is sequestered inside the cell, in the membrane of intracellular vesicles. Insulin triggers a biochemical cascade that causes the vesicle membranes to fuse with the surface membrane (exocytosis), exposing the glucose transporter to the ECF. The transporter then gets busy and ‘carries’ glucose inside. When insulin subsides, the transporter molecules are reinternalized (endocytosis).
Primary active transporters
like the Na/K pump, derive their energy directly from the splitting of ATP. There are no other ubiquitous primary active transporters in the plasma membrane of cells. Some specialized cells have them, and you will study them later. For example, the cells in the stomach that secrete acid, and certain cells in the kidney geared for excreting protons from the body possess proton pumps in their plasma membranes that rely directly on ATP for their energy source. Inside cells (as opposed to the surface membrane) there are other primary active transporters. One pumps protons into intracellular membrane-bound organelles (endosomes, vesicles, lysosomes). Another pumps calcium ions into membrane-bound compartments. Inside mitochondria is a very special proton pump (the F1-ATPase) that, when running backwards, lets protons leak across a membrane and synthesizes, rather than hydrolyzes ATP.
Secondary active transport
the mechanism by which most substances are pumped. In this case, the energy to do the direct work of pumping comes not from metabolism (ATP), but from a secondary source. Usually this energy source is the ‘downhill leak’ of Na+ into the cell. For example, cells can accumulate amino acids against their energy gradients. This active uptake is dependent on external Na+; if external Na+ is removed, amino acid uptake is abolished. Conversely, removing the amino acid reduces the entry of Na+. The carrier ingeniously captures the energy released by the inward leak of Na+ and instead of letting it escape as heat, uses it to pump the amino acid into the cell. There are two basic types of secondary active transporters, those that move different solute species in the same direction (cotransport), and those that move solute in opposite directions (antiport, or exchange). Secondary active transporters do not necessarily always run in the same direction. They will always tap the bigger leak to drive the smaller pump. Consequently, they can reverse direction sometimes. One of the most important examples of this is the sodium-calcium exchanger, which reverses direction in heart muscle cells every time the heart beats (more on this later). All secondary transport mechanisms depend ultimately on the Na+/K+ pump (and therefore on ATP). For example, if the Na+/K+ pump is blocked, cells fill up with Na+, and thus the Na+ electrochemical gradient is reduced. Because this is the energy source for secondary active transport, all of these transport mechanisms suffer. Some secondary active transporters are electrogenic, in that one cycle produces a net charge transfer across the membrane. For example, Na/amino acid transporters are electrogenic, because one cycle transfers a net positive charge (Na+) into the cell. Other secondary active transporters are not electrogenic; an example is the Na/K/2Cl cotransporter, which each cycle moves one sodium ion, one potassium ion, and two chloride ions into the cell. The main feature of electrogenic secondary active transporters is that their activity is governed by the membrane potential. Electrically silent transporters could not care less about membrane potential.
Calcium transport
There is a huge electrochemical gradient for calcium ions across cell membranes. In fact, no other ion is further from equilibrium than calcium. The extracellular (ionized) calcium concentration (about 1 mM) is nearly 10,000 times greater than the intracellular concentration (about 0.0002 mM, or 200 nM); thus its concentration gradient is inward. And the electrical gradient is also inward, of course, because the ICF is electrically negative and calcium is positively charged. From the Nernst equation, ECa is calculated to be about +111 mV (ECa = (60/2)*log(1/0.0002) = +111 mV). Thus, given the opportunity (i.e., an open calcium channel), Ca++ ions will always leak into cells, so there must be a pump to extrude them. The Na/Ca exchanger’s main job is to pump calcium ions out of the cell. The inward leak of sodium ions provides the energy source. The Na/Ca exchange pump takes on a special significance in the heart, where it actually switches direction during each heartbeat. (You will study muscle contraction shortly, and will learn that calcium ions are necessary for contraction.) At rest (while the ventricles are refilling with blood during diastole), the exchanger runs forward, pumping Ca++ out (keeping the muscle relaxed) as Na+ leaks in. When the ventricles contract (systole), the exchanger switches direction, letting Ca++ leak into the cell, where it strengthens the force of contraction. The direction of Ca++ movement is controlled by the value of the membrane potential; when Vm is more negative than about -60 mV, the sodium leak rules, and drives the outward pumping of calcium; when Vm is more positive than -60, the pump reverses direction, and calcium leaks in (pumping sodium out).
Digitalis
For centuries it has been known that an extract of the beautiful purple foxglove can help a weak heart beat stronger. Digitalis (and related drugs) exert their action by acting directly on, not the Na/Ca exchanger, but the Na/K pump! In fact, digitalis blocks the Na/K pump. How does this lead to an increase in the strength of contraction of heart muscle? Blocking the Na/K pump of course allows intracellular sodium ion concentration to increase. In turn, this reduces the energy available to all sodium-driven secondary active transporters, including the Na/Ca exchanger. Thus, digitalis indirectly inhibits the Na/Ca exchanger, allowing intracellular calcium ion concentration to rise, which increases cardiac contractility.
Hydrogen ions
are also pumped out of most cells by a Na+/H+ exchange carrier, which operates under the same principles as the Na/Ca exchanger. Protons are harder to study than UFO’s, because they capriciously vanish and reappear as they bind to and unbind from various buffers. Typically, only about 1 in a million protons is free, as H+; the rest are hiding, bound to buffers. The free concentration of protons in the ICF is about 100 nM (pH=7.0); in the ECF it’s even lower, about 40 nM (pH=7.4). From the Nernst equation, EH = -24 mV. That means in cells with membrane potentials more negative than -24 mV (most cells), H+ must be pumped out of the cell. The mechanism is a secondary active transport system, in which the inward leak of Na+ drives the outward pumping of H+.
Chloride ions
are pumped into some cells by a secondary active transport process (Na/K/2Cl cotransporter). As a result, ECl moves in a positive direction, away from the resting membrane potential.
H+/K+ exchanger’
There are several clinical situations that suggest the presence of a system that will exchange K+ for H+, and vice versa. For example, infusing K+ causes acidemia (the K+ is taken up by cells ‘in exchange’ for H+), and infusing acid causes hyperkalemia (elevated (hyper-) potassium (-kal- for Latin kalium) in the blood (-emia)). While it is conceptually simple (and useful) to think in terms of an H/K exchanger, the reality is that such a transporter probably does not exist. Rather, the process evidently involves different transporters, perhaps working in pairs in parallel, the upshot being hydrogen/potassium exchange. For example, hyperkalemia will cause extra K+ uptake via the Na/K pump. Hyperkalemia also will depolarize cells (by shifting EK in a positive direction), and the change in membrane potential can affect the rate of activity of electrogenic transporters. One such transporter, which transports 3 bicarbonate ions and one sodium ion from the ICF to the ECF, is inhibited by depolarization. Thus, the reduction in activity will reduce bicarbonate extrusion. Because bicarbonate is a base, its slower extrusion will cause acidemia.
CYTOSKELETON
The cytoskeleton provides cell shape, mechanical strength, the structures needed for locomotion, support for the plasma membrane, the scaffold for the spatial organization of organelles, and the means for intracellular transport of organelles and other cargo. The cytoskeleton is formed by three different families of proteins that assemble to form large filamentous or tubular, non-covalent polymers, each with distinct mechanical properties, dynamics and functions. The three types of cytoskeletal element are: microfilaments, microtubules, and intermediate filaments. Microfilaments (synonymous with actin cytoskeleton)
Microtubules (MTs)
are tubular (or hollow-fiber) structures, up to many μm long, with an outer diameter of 25nm. They are flexible but not very resistant to stretching. They function primarily as scaffolds for the spatial organization of organelles in the cell, for organelle movement, and for the movement of cilia and flagella. They typically have one end attached to a centrosome, also known as perinuclear microtubule organizing center (MTOC).
tubulin
The building blocks of MTs are heterodimers of the protein tubulin (α and β). Each tubulin has a binding site for GTP. The GTP bound to a tubulin is a trapped at the tubulin interface of the dimer and is not hydrolized. It is a constant heterodimer component. The GTP bound to β tubulin can be hydrolized and is exchangeable. There are many isoforms. the main difference is the tail, which is the charged domain These tails are not part of the conserved tubulin fold; instead, they protrude from the surface of polymerized microtubules and are thus readily accessible for interacting molecules. To form microtubules, the dimers of α- and β-tubulin bind to GTP and assemble onto the (+) ends of microtubules while in the GTP-bound state. The β-tubulin subunit is exposed on the plus end of the microtubule while the α-tubulin subunit is exposed on the minus end. After the dimer is incorporated into the microtubule, the molecule of GTP bound to the β-tubulin subunit eventually hydrolyzes into GDP through inter-dimer contacts along the microtubule protofilament. Whether the β-tubulin member of the tubulin dimer is bound to GTP or GDP influences the stability of the dimer in the microtubule. Dimers bound to GTP tend to assemble into microtubules, while dimers bound to GDP tend to fall apart; thus, this GTP cycle is essential for the dynamic instability of the microtubule.
protofilaments
MTs are hollow cylinders composed of 13 parallel “protofilaments” consisting of alternating α and β subunits, i.e., of chains of heterodimers in which the top of one b tubulin interacts with the bottom of the a tubulin of the next heterodimer. Laterally, the protofilaments are slightly displaced (forming a spiral), but the interactions are mostly α−α and β−β. Because intermolecular interactions are strong within the assembled tubule, there is little exchange of dimers within. The ends, however, exhibit (controlled) rapid assembly and disassembly. Because of their architecture, MTs exhibit on one end α tubulin, on the other β tubulin and, thus, have polarity.
treadmilling
Because GTP-bearing β subunits favor polymerization, that end of the tubule, known as plus-end, is the one that predominantly grows. The opposite end, or minus-end, tends to be disassembling or shrinking. As GTP- containing dimers become incorporated more deeply in MTs, GTP is hydrolized to GDP, which weakens the tubulin interaction in the protofilament. This results ultimately in a “treadmilling” phenomenon (MT growth at the plus-end, MT disassembly at the minus-end).
dynamic instability
However in the response to a particular cellular activity, the plus-end may loose its GTP-rich cap, which causes rapid shrinkage from the plus-end until GTP-containing dimers are added back. This phenomenon of rapid MT dynamics is known as “dynamic instability”. MT associating proteins can control MT stability by either protecting or removing GTP-cap. MT capping proteins that bind to the ends of microtubules, usually increase their stability. In contrast, microtubule severing proteins (such as spastin, katanin) increase microtubule instability by exposing GDP- rich parts of microtubules.
perinuclear microtubule organizing center (MTOC)
a structure found in eukaryotic cells from which microtubules emerge. MTOCs have two main functions: the organization of eukaryotic flagella and cilia and the organization of the mitotic and meiotic spindle apparatus, which separate the chromosomes during cell division. Most animal cells contain a single MTOC, called the centrosome and located near the nucleus. It is composed of a pair of centrioles embedded in a matrix and of nucleation sites for MTs. These nucleation sites, consisting of rings of γ-tubulin, can initiate MT polymerization, and the minus-ends of MTs are anchored in them.
centrioles
a cylindrical cell structure composed mainly of a protein called tubulin that is found in most eukaryotic cells. are a pair of cylindrical structures arranged at right angles to each other and consist of short modified tubules plus accessory proteins. Centrioles also form the basal bodies of cilia and flagella. During each cell cycle the centrosome duplicates and splits into equal parts, each containing a centriole pair. The centrosomes move to opposite ends of the cell at the onset of mitosis, and the MTs growing between them form the mitotic spindle for the separation of chromosomes.
Drugs that modify MT polymerization dynamics
have been isolated from plants. Colchicine (from the fall-flowering, crocus-like Colchicum autumnale) inhibits MT polymerization. Vinblastine and vincristine also are MT polymerization blockers, derived from the Madagascar periwinkle (Vinca rosea). Paclitaxel (Taxol®) was first isolated from the Pacific yew tree (Taxus brevifolia) and also binds to MTs, but it stabilizes them, which causes tubule and tubulin aggregates. These compounds and their derivatives block mitosis and, thus, are of great interest for cancer treatment.
Microtubule Functions
a) Scaffold for organelle positioning in the cell: Various organelles, such as ER and Golgi, are anchored to MTs. b) Intracellular transport. c) Cell division. d) Cilia formation and movement
MTs role in Intracellular transport
Many organelles (vesicles, mitochondria) travel long distances within cells, using MTs as “tracks”. This is possible in conjunction with microtubule motor proteins. These proteins can transform the energy from ATP hydrolysis into motion (or walking) along MTs (molecular motors). They contain a cargo-binding domain and a “head” region or motor domain that hydrolyzes ATP and reversibly binds to MTs. Coordinated with ATP hydrolysis, the motor domain goes through a mechanochemical cycle of MT binding, conformational change, MT release, conformational relaxation, and MT re-binding. This moves motor protein and cargo along the MT in a stepwise fashion. These principles are shared with the myosins discussed further below. Two different classes of MT motors exist, kinesins, which move cargo toward the plus-end, and dyneins, which move cargo toward the minus-end.
MTs role in Cell division
The mitotic spindle is constructed from MTs and associated proteins and serves to segregate the replicated chromosomes during mitosis. Three types of MTs can be distinguished: “astral MTs” that radiate out from the centrosomes; “kinetochore MTs” that are attached to the kinetochore formed at the centromere of each duplicated chromosome; and “overlap MTs” that interdigitate at the equator of the spindle. In all cases, MT plus-ends point away from the centrosomes (or spindle poles). Multimeric plus-end-directed, kinesin-like motor proteins bind to overlap MTs from opposite poles. These motors, as well as the elongation of overlap MTs, cause the spindle to grow and the centrosomes to become more distant. This is enhanced by pulling forces transmitted by astral MTs. At the same time, minus-end directed motors, accompanied by kinetochore MT shortening, separate daughter chromosomes and move them along MTs to the centrosomes.
kinesins
Kinesins are motor proteins that transport such cargo by walking unidirectionally along microtubule tracks hydrolysing one molecule of adenosine triphosphate (ATP) at each step. It was thought that ATP hydrolysis powered each step, the energy released propelling the head forwards to the next binding site. However, it has been proposed that the head diffuses forward and the force of binding to the microtubule is what pulls the cargo along. Motor proteins travel in a specific direction along a microtubule. This is because the microtubule is polar and the heads only bind to the microtubule in one orientation, while ATP binding gives each step its direction through a process known as neck linker zippering. Most kinesins walk towards the plus end of a microtubule which, in most cells, entails transporting cargo from the centre of the cell towards the periphery. This form of transport is known as anterograde transport/orthrograde transport.
dyneins
The force-generating ATPase activity of each dynein heavy chain is located in its large doughnut-shaped “head”, which is related to other AAA proteins, while two projections from the head connect it to other cytoplasmic structures. One projection, the coiled-coil stalk, binds to and “walks” along the surface of the microtubule via a repeated cycle of detachment and reattachment. The other projection, the extended tail (also called “stem”), binds to the intermediate and light chain subunits which attach the dynein to its cargo. The alternating activity of the paired heavy chains in the complete cytoplasmic dynein motor enables a single dynein molecule to transport its cargo by “walking” a considerable distance along a microtubule without becoming completely detached. Several mutations in the dyneim motor forms a subclass of “primary ciliary dyskinesia syndromes”. Due to a mutation the outer dynein arms in cilia and flagella are missing. When explaining the syndrome, note that photoreceptor outer segments are modified monocilia, that such cilia may operate as chemosensors (anosmia; probable explanation for cyst formation in developing kidney), and that establishment of left/right identity requires normal function of monocilia on nodal cells during gastrulation
Mitotic spindle
A notable structure involving microtubules is the mitotic spindle, used by most eukaryotic cells to segregate their chromosomes correctly during cell division. The mitotic spindle includes the spindle microtubules, microtubule-associated proteins (MAPs), and the MTOC. The microtubules originate in the MTOC and fan out into the cell; each cell has two MTOCs, as shown in the diagram. The process of mitosis is facilitated by three main subgroups of microtubules, known as astral, overlap and kinetochore microtubules.
astral microtubule
a microtubule originating from the MTOC that does not connect to a chromosome. Astral microtubules instead interact with the cytoskeleton near the cell membrane and function in concert with specialized dynein motors, which pull the MTOC toward the cell membrane, thus assisting in correct positioning and orientation of the entire apparatus.
Kinetochore microtubules
directly connect to the chromosomes, at the kinetochores. To clarify the terminology, each chromosome has two chromatids, and each chromatid has a kinetochore; the two kinetochores are linked. The complex created by the two kinetochores on a chromosome is called the centromere.
overlap microtubules
extend from one MTOC and intertwine with the microtubules from the other MTOC; motor proteins then make them push against each other and assist in the separation of the two daughter cells. Keinesin attached to both MTs will help seperate the centrosome by having two motor heads attached to each MT and walk towards + end.
MTs role in Cilia formation and movement
Cilia and flagella are wide-spread, hair-like cellular appendages that have a uniform diameter and contain a MT core, the axoneme. Flagella are long and serve to propel sperm by their undulating motion. Cilia are shorter and tend to occur in large numbers on the apical surface of various epithelial cells, especially those of the respiratory tract. By beating with a whip-like motion (in a staggered pattern across the cell surface) they move fluids over the surfaces of cells. In the respiratory tract, this serves to move dust particles, bacteria and mucus towards the mouth for elimination.
Intermediate filaments (IFs)
are rope-like, fibrous structures of about 10nm diameter. Unlike MTs and microfilaments, they are an invention of metazoans. IFs are prominent in cells exposed to mechanical stress. The main function of IFs is to provide intracellular mechanical support. IFs fall into two categories: cytoplasmic IFs and nuclear lamins. Several cell-type-specific cytoplasmic IF proteins exist: keratins, vimentins and vimentin-related proteins (including glial fibrillary acidic protein), and neurofilament proteins. In other words, IF proteins are far more heterogeneous than those of microfilaments and MTs. All IF proteins are elongated molecules with an extended central α-helical domain that forms a parallel coiled-coil with another monomer. Pairs of dimers then associate in anti-parallel fashion to form staggered tetramers. These tetramers are the soluble subunits that participate in IF polymerization. They are not polarized. IF polymerization is nucleotide-independent. Tetramers assemble into protofilaments, and eight such protofilaments pack together laterally to form the IF. Thus, at all levels IF cross- sections contain 32 individual α-helical coils that provide great tensile strength. IFs often are anchored in intercellular junctions. Is not polar therefore is more stable
keratins
only in epithelia. a family of about 50 proteins, are dominant components of the epidermis and its appendages, providing mechanical strength. a family of fibrous structural proteins. Keratin is the key structural material making up the outer layer of human skin. It is also the key structural component of hair and nails. Keratin monomers assemble into bundles to form intermediate filaments, which are tough and form strong unmineralized tissues found in reptiles, birds, amphibians, and mammals. Keratin mutations may interfere with filament assembly. The resulting epidermis is highly sensitive to mechanical stress and blisters easily, causing a severe disorder called epidermolysis bullosa simplex. Mutations in some of the associated proteins (e.g., those that anchor the filaments in desmosomes) may result in similar clinical syndromes. It is a heterodimer (acidic and basic keratins). Can be a marker for cancer cells because it will maintain the type of keratin expressed by the cell that it derieved from. hepatocytes only have one set of keratins, therefore keratin mutations in these proteins will lead to liver failure. Other types of cells have more than one set and therefore can compensate better with such mutations.
vimentins
are in connective tissue, muscle cells, and neuroglial cells. Vimentins are present in a majority of cell types. a type III intermediate filament (IF) protein that is expressed in mesenchymal cells. Vimentin plays a significant role in supporting and anchoring the position of the organelles in the cytosol. Vimentin is attached to the nucleus, endoplasmic reticulum, and mitochondria, either laterally or terminally
neurofilament
(three types) co-assemble to form neurofilaments, which are found in high concentration in vertebrate axons. Neurofilament abundance appears to control axonal diameter. They are a major component of the neuronal cytoskeleton, and are believed to function primarily to provide structural support for the axon and to regulate axon diameter. Mutations in the light chain may interfere with axonal transport of neurofilament subunits and cause a peripheral neuropathy called Charcot-Marie-Tooth syndrome. Abnormal neurofilament assembly seems to be involved in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig’s Disease).
nuclear lamins
are filamentous proteins that form a stabilizing meshwork lining the inner membrane of the nuclear envelope to provide anchorage for chromosomes and nuclear pores. Mutations in lamins can result in nuclear instability. Lamin mutations are linked to various progeria syndroms.
Glial fibrillary acidic protein (GFAP
the IF protein characteristic of astrocytes in the CNS. It is involved in many important CNS processes, including cell communication and the functioning of the blood brain barrier. Glial scarring is a consequence of several neurodegenerative conditions, as well as injury that severs neural material. The scar is formed by astrocytes interacting with fibrous tissue to re-establish the glial margins around the central injury core and is partially caused by up-regulation of GFAP. Therefore, GFAP IFs are abundant in connection with inflammatory and/or degenerative processes in the brain. For example, Alzheimer plaques are surrounded by GFAP-rich reactive astrocytes.
epidermolysis bullosa simplex
a disorder resulting from mutations in the genes encoding keratin 5 or keratin 14. Blister formation of EBS occurs at the dermoepidermal junction. Sometimes EBS is called epidermolytic
Microtubules as drug targets
MT toxins block mitosis and, thus, are important therapeutic tools for cancer treatment. However, they may adversely affect other MT functions, such as axoplasmic transport. Thus, side effects (e.g. peripheral neuropathy) may be serious.
Microfilaments
and associated proteins (actin cytoskeleton) form the third structural component of the cytoskeleton. They are structurally and functionally distinct from microtubules (MTs) and intermediate filaments. Microfilaments are essential for amoeboid motility. Microfilaments are filamentous polymers (~7 nm diameter) of actin monomers (“globular actin” = G-actin). In the presence of divalent cations and ATP, G-actin assembles to form two- stranded, helical filaments (F-actin). Most of these filaments contain additional actin-binding proteins. Microfilaments (MFs) are critical for cell shape, movement and polarity.
Actin
a globular multi-functional protein that forms microfilaments. It is found in all eukaryotic cells (the only known exception being nematode sperm), where it may be present at concentrations of over 100 μM. An actin protein’s mass is roughly 42-kDa and it is the monomeric subunit of two types of filaments in cells: microfilaments, one of the three major components of the cytoskeleton, and thin filaments, part of the contractile apparatus in muscle cells. Most obvious are actin’s roles in muscle contraction and amoeboid cell movement; however, these are not the only microfilament functions. MFs, just like MTs, are polarized (due to orientation in the filament and molecular asymmetry of the subunits). Some 60 accessory proteins - a huge number - participate in the regulation of polymerization and disassembly. These include proteins that bind G-actin, others that stabilize, crosslink, sever or cap F-actin, and proteins that enable F-actin branching to form MF networks.
G-actin
This structure represents the “ATPase fold”, which is a centre of enzymatic catalysis that binds ATP and Mg2+ and hydrolyzes the former to ADP plus phosphate. This fold is a conserved structural motif that is also found in other proteins that interact with triphosphate nucleotides such as hexokinase (an enzyme used in energy metabolism) or in Hsp70 proteins (a protein family that play an important part in protein folding).[21] G-actin is only functional when it contains either ADP or ATP in its cleft but the form that is bound to ATP predominates in cells when actin is present in its free state
F-actin
The F-actin polymer is considered to have structural polarity due to the fact that all the microfilament’s subunits point towards the same end. This gives rise to a naming convention: the end that possesses an actin subunit that has it’s ATP binding site exposed is called the “(-) end”, while the opposite end where the cleft is directed at a different adjacent monomer is called the “(+) end”
phalloidin
extracted from the highly toxic fungus Amanita phalloides (“death cap”), which binds to and stabilizes F-actin (causing a net increase in actin polymerization). Actin filament nucleation typically occurs at the plasma membrane, which accounts for the high density of MFs in the cell periphery.
Arp2/3
a seven-subunit protein that plays a major role in the regulation of the actin cytoskeleton. It is a major component of the actin cytoskeleton and is found in most actin cytoskeleton-containing eukaryotic cells. Two of its subunits, the Actin-Related Proteins ARP2 and ARP3 closely resemble the structure of monomeric actin and serve as nucleation sites for new actin filaments. The complex binds to the sides of existing (“mother”) filaments and initiates growth of a new (“daughter”) filament at a distinctive 70 degree angle from the mother. Branched actin networks are created as a result of this nucleation of new filaments. The ARP (or Arp2/3) complex nucleates MF polymerization from the minus-end, allowing rapid elongation at the plus-end. Arp2/3 activation results in branched actin filaments (required for lamellipodia formation).
nucleation
the first step in the formation of either a new thermodynamic phase or a new structure via self-assembly or self-organisation. Nucleation is typically defined to be the process that determines how long we have to wait before the new phase or self-organised structure, appears. Nucleation with microfilaments is catalyzed by several regulatory proteins. One of them is a protein complex that includes two “actin-related proteins” (ARPs). Second regulator of actin nucleation regulator is forming. Activation of forming leads to parallel actin bundle formation (mirovilli, filopodia, actomyosin ring).
Actin and epithelial cell polarity
Actin plays a key role in polarization of epithelial cells. One of the most important functions of actin is anchoring proteins that are involved in Tight junction (TJ) and Adherens junction (AJ) formation. An unusual type of actin anchoring is observed in brush border microvilli: A tight MF bundle forms the core of these microvilli. All actin plus-ends are anchored in the apical protein cap of the microvillus. Actin bundles are held together by the cross-linking proteins villin and fimbrin, and bundles are linked laterally to the plasma membrane by myosin-I. Loss of microvilli is observed in microvilli inclusion disease.
Adherens junction (AJ)
are protein complexes that occur at cell–cell junctions in epithelial and endothelial tissues,[2] usually more basal than tight junctions. An adherens junction is defined as a cell junction whose cytoplasmic face is linked to the actin cytoskeleton. Decreased association of AJ proteins (cadherins and catenins) with actin leads to internalization of candherins and loss of cell-cell adhesion, the step that is a prerequisite for epithelial-to-mesenchimal (EMT) transition and cancer formation.
epithelial-to-mesenchimal (EMT) transition
a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells; these are multipotent stromal cells that can differentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis for cancer progression. Epithelial and mesenchymal cells differ in phenotype as well as function. Epithelial cells are closely connected to each other by tight junctions, gap junctions and adherens junctions, have an apico-basal polarity, polarization of the actin cytoskeleton and are bound by a basal lamina at their basal surface. Mesenchymal cells, on the other hand, lack this polarization, have a spindle-shaped morphology and interact with each other only through focal points. Epithelial cells express high levels of E-cadherin, whereas mesenchymal cells express those of N-cadherin, fibronectin and vimentin. Thus, EMT entails profound morphological and phenotypic changes to a cell. Based on the biological context, EMT has been categorized into 3 types - developmental (Type I), fibrosis and wound healing (Type II), and cancer (Type III).
microvilli inclusion disease
Microvillus inclusion disease is a condition characterized by chronic, watery, life-threatening diarrhea typically beginning in the first hours to days of life. Rarely, the diarrhea starts around age 3 or 4 months. Food intake increases the frequency of diarrhea. a rare genetic disorder of the small intestine that is inherited in an autosomal recessive pattern. Mutations in the MYO5B gene cause microvillus inclusion disease. The MYO5B gene provides instructions for making a protein called myosin Vb. This protein helps to determine the position of various components within cells (cell polarity). Cell loses polartiy. Myosin Vb also plays a role in moving components from the cell membrane to the interior of the cell for recycling. Don’t for ARP2/3 structures.
cadherins
a class of type-1 transmembrane proteins. They play important roles in cell adhesion, forming adherens junctions to bind cells within tissues together. They are dependent on calcium (Ca2+) ions to function, hence their name.
