Spindle apparatus

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https://en.wikipedia.org/wiki/Spindle_apparatus

Micrograph showing condensed chromosomes in blue, kinetochores in pink, and microtubules in green during metaphase of mitosis

In cell biology, the spindle apparatus is the cytoskeletal structure of eukaryotic cells that forms during cell division to separate sister chromatids between daughter cells. It is referred to as the mitotic spindle during mitosis, a process that produces genetically identical daughter cells, or the meiotic spindle during meiosis, a process that produces gametes with half the number of chromosomes of the parent cell.

Besides chromosomes, the spindle apparatus is composed of hundreds of proteins. Microtubules comprise the most abundant components of the machinery.

Spindle structure

This diagram depicts the organization of a typical mitotic spindle found in animal cells. Chromosomes are attached to kinetochore microtubules via a multiprotein complex called the kinetochore. Polar microtubules interdigitate at the spindle midzone and push the spindle poles apart via motor proteins. Astral microtubules anchor the spindle poles to the cell membrane. Microtubule polymerization is nucleated at the microtubule organizing center.

Attachment of microtubules to chromosomes is mediated by kinetochores, which actively monitor spindle formation and prevent premature anaphase onset. Microtubule polymerization and depolymerization dynamic drive chromosome congression. Depolymerization of microtubules generates tension at kinetochores; bipolar attachment of sister kinetochores to microtubules emanating from opposite cell poles couples opposing tension forces, aligning chromosomes at the cell equator and poising them for segregation to daughter cells. Once every chromosome is bi-oriented, anaphase commences and cohesin, which couples sister chromatids, is severed, permitting the transit of the sister chromatids to opposite poles.

The cellular spindle apparatus includes the spindle microtubules, associated proteins, which include kinesin and dynein molecular motors, condensed chromosomes, and any centrosomes or asters that may be present at the spindle poles depending on the cell type. The spindle apparatus is vaguely ellipsoid in cross section and tapers at the ends. In the wide middle portion, known as the spindle midzone, antiparallel microtubules are bundled by kinesins. At the pointed ends, known as spindle poles, microtubules are nucleated by the centrosomes in most animal cells. Acentrosomal or anastral spindles lack centrosomes or asters at the spindle poles, respectively, and occur for example during female meiosis in most animals. In this instance, a Ran GTP gradient is the main regulator of spindle microtubule organization and assembly. In fungi, spindles form between spindle pole bodies embedded in the nuclear envelope, which does not break down during mitosis.

Microtubule-associated proteins and spindle dynamics

The dynamic lengthening and shortening of spindle microtubules, through a process known as dynamic instability determines to a large extent the shape of the mitotic spindle and promotes the proper alignment of chromosomes at the spindle midzone. Microtubule-associated proteins (MAPs) associate with microtubules at the midzone and the spindle poles to regulate their dynamics. γ-tubulin is a specialized tubulin variant that assembles into a ring complex called γ-TuRC which nucleates polymerization of α/β tubulin heterodimers into microtubules. Recruitment of γ-TuRC to the pericentrosomal region stabilizes microtubule minus-ends and anchors them near the microtubule-organizing center. The microtubule-associated protein Augmin acts in conjunction with γ-TURC to nucleate new microtubules off of existing microtubules.

The growing ends of microtubules are protected against catastrophe by the action of plus-end microtubule tracking proteins (+TIPs) to promote their association with kinetochores at the midzone. CLIP170 was shown to localize near microtubule plus-ends in HeLa cells and to accumulate in kinetochores during prometaphase. Although how CLIP170 recognizes plus-ends remains unclear, it has been shown that its homologues protect against catastrophe and promote rescue, suggesting a role for CLIP170 in stabilizing plus-ends and possibly mediating their direct attachment to kinetochores. CLIP-associated proteins like CLASP1 in humans have also been shown to localize to plus-ends and the outer kinetochore as well as to modulate the dynamics of kinetochore microtubules (Maiato 2003). CLASP homologues in Drosophila, Xenopus, and yeast are required for proper spindle assembly; in mammals, CLASP1 and CLASP2 both contribute to proper spindle assembly and microtubule dynamics in anaphase. Plus-end polymerization may be further moderated by the EB1 protein, which directly binds the growing ends of microtubules and coordinates the binding of other +TIPs.

Opposing the action of these microtubule-stabilizing proteins are a number of microtubule-depolymerizing factors which permit the dynamic remodeling of the mitotic spindle to promote chromosome congression and attainment of bipolarity. The kinesin-13 superfamily of MAPs contains a class of plus-end-directed motor proteins with associated microtubule depolymerization activity including the well-studied mammalian MCAK and Xenopus XKCM1. MCAK localizes to the growing tips of microtubules at kinetochores where it can trigger catastrophe in direct competition with stabilizing +TIP activity. These proteins harness the energy of ATP hydrolysis to induce destabilizing conformational changes in protofilament structure that cause kinesin release and microtubule depolymerization. Loss of their activity results in numerous mitotic defects. Additional microtubule destabilizing proteins include Op18/stathmin and katanin which have roles in remodeling the mitotic spindle as well as promoting chromosome segregation during anaphase.

The activities of these MAPs are carefully regulated to maintain proper microtubule dynamics during spindle assembly, with many of these proteins serving as Aurora and Polo-like kinase substrates.

Organizing the spindle apparatus

In the centrosome-mediated "search and capture" model (left), microtubules nucleated from centrosomes contact chromosomes by chance and become stabilized at kinetochores to form the spindle. In the chromatin-mediated "self-organization" model (right), microtubules are nucleated around the vicinity of mitotic chromatin and organized into a bipolar array by motor proteins.

In a properly formed mitotic spindle, bi-oriented chromosomes are aligned along the equator of the cell with spindle microtubules oriented roughly perpendicular to the chromosomes, their plus-ends embedded in kinetochores and their minus-ends anchored at the cell poles. The precise orientation of this complex is required to ensure accurate chromosome segregation and to specify the cell division plane. However, it remains unclear how the spindle becomes organized. Two models predominate the field, which are synergistic and not mutually exclusive. In the search-and-capture model, the spindle is predominantly organized by the poleward separation of centrosomal microtubule organizing centers (MTOCs). Spindle microtubules emanate from centrosomes and 'seek' out kinetochores; when they bind a kinetochore they become stabilized and exert tension on the chromosomes. In an alternative self assembly model, microtubules undergo acentrosomal nucleation among the condensed chromosomes. Constrained by cellular dimensions, lateral associations with antiparallel microtubules via motor proteins, and end-on attachments to kinetochores, microtubules naturally adopt a spindle-like structure with chromosomes aligned along the cell equator.

Centrosome-mediated "search-and-capture" model

In this model, microtubules are nucleated at microtubule organizing centers and undergo rapid growth and catastrophe to 'search' the cytoplasm for kinetochores. Once they bind a kinetochore, they are stabilized and their dynamics are reduced. The newly mono-oriented chromosome oscillates in space near the pole to which it is attached until a microtubule from the opposite pole binds the sister kinetochore. This second attachment further stabilizes kinetochore attachment to the mitotic spindle. Gradually, the bi-oriented chromosome is pulled towards the center of the cell until microtubule tension is balanced on both sides of the centromere; the congressed chromosome then oscillates at the metaphase plate until anaphase onset releases cohesion of the sister chromatids.

In this model, microtubule organizing centers are localized to the cell poles, their separation driven by microtubule polymerization and 'sliding' of antiparallel spindle microtubules with respect to one another at the spindle midzone mediated by bipolar, plus-end-directed kinesins. Such sliding forces may account not only for spindle pole separation early in mitosis, but also spindle elongation during late anaphase.

Chromatin-mediated self-organization of the mitotic spindle

In contrast to the search-and-capture mechanism in which centrosomes largely dictate the organization of the mitotic spindle, this model proposes that microtubules are nucleated acentrosomally near chromosomes and spontaneously assemble into anti-parallel bundles and adopt a spindle-like structure. Classic experiments by Heald and Karsenti show that functional mitotic spindles and nuclei form around DNA-coated beads incubated in Xenopus egg extracts and that bipolar arrays of microtubules are formed in the absence of centrosomes and kinetochores. Indeed, it has also been shown that laser ablation of centrosomes in vertebrate cells inhibits neither spindle assembly nor chromosome segregation. Under this scheme, the shape and size of the mitotic spindle are a function of the biophysical properties of the cross-linking motor proteins.

Chromatin-mediated microtubule nucleation by the Ran GTP gradient

The guanine nucleotide exchange factor for the small GTPase Ran (Regulator of chromosome condensation 1 or RCC1) is attached to nucleosomes via core histones H2A and H2B. Thus, a gradient of GTP-bound Ran is generated around the vicinity of mitotic chromatin. Glass beads coated with RCC1 induce microtubule nucleation and bipolar spindle formation in Xenopus egg extracts, revealing that the Ran GTP gradient alone is sufficient for spindle assembly. The gradient triggers release of spindle assembly factors (SAFs) from inhibitory interactions via the transport proteins importin β/α. The unbound SAFs then promote microtubule nucleation and stabilization around mitotic chromatin, and spindle bipolarity is organized by microtubule motor proteins.

Regulation of spindle assembly

Spindle assembly is largely regulated by phosphorylation events catalyzed by mitotic kinases. Cyclin dependent kinase complexes (CDKs) are activated by mitotic cyclins, whose translation increases during mitosis. CDK1 (also called CDC2) is considered the main mitotic kinase in mammalian cells and is activated by Cyclin B1. Aurora kinases are required for proper spindle assembly and separation. Aurora A associates with centrosomes and is believed to regulate mitotic entry. Aurora B is a member of the chromosomal passenger complex and mediates chromosome-microtubule attachment and sister chromatid cohesion. Polo-like kinase, also known as PLK, especially PLK1 has important roles in the spindle maintenance by regulating microtubule dynamics.

Mitotic chromosome structure

By the end of DNA replication, sister chromatids are bound together in an amorphous mass of tangled DNA and protein. Mitotic entry triggers a dramatic reorganization of the duplicated genome, resulting in sister chromatids that are disentangled and separated from one another. Chromosomes also shorten in length, up to 10,000-fold in animal cells, in a process called condensation. Condensation begins in prophase and chromosomes are maximally compacted into rod-shaped structures by the time they are aligned in the middle of the spindle at metaphase. This gives mitotic chromosomes the classic "X" shape seen in karyotypes, with each condensed sister chromatid linked along their lengths by cohesin proteins and joined, often near the center, at the centromere.

While these dynamic rearrangements are vitally important to ensure accurate and high-fidelity segregation of the genome, our understanding of mitotic chromosome structure remains largely incomplete. A few specific molecular players have been identified, however: Topoisomerase II uses ATP hydrolysis to catalyze decatenation of DNA entanglements, promoting sister chromatid resolution. Condensins are 5-subunit complexes that also use ATP-hydrolysis to promote chromosome condensation. Experiments in Xenopus egg extracts have also implicated linker Histone H1 as an important regulator of mitotic chromosome compaction.

Mitotic spindle assembly checkpoint

The completion of spindle formation is a crucial transition point in the cell cycle called the spindle assembly checkpoint. If chromosomes are not properly attached to the mitotic spindle by the time of this checkpoint, the onset of anaphase will be delayed. Failure of this spindle assembly checkpoint can result in aneuploidy and may be involved in aging and the formation of cancer.

Spindle apparatus orientation

Cartoon of the dividing epithelium cell surrounded by epithelium tissue. Spindle apparatus rotates inside the cell. The rotation is a result of astral microtubules pulling towards tri-cellular-junctions (TCJ), signaling centers localized at the regions where three cells meet.

Cell division orientation is of major importance for tissue architecture, cell fates and morphogenesis. Cells tend to divide along their long axis according to the so-called Hertwig rule. The axis of cell division is determined by the orientation of the spindle apparatus. Cells divide along the line connecting two centrosomes of the spindle apparatus. After formation, the spindle apparatus undergoes rotation inside the cell. The astral microtubules originating from centrosomes reach the cell membrane where they are pulled towards specific cortical clues. In vitro, the distribution of cortical clues is set up by the adhesive pattern. In vivo polarity cues are determined by localization of Tricellular junctions localized at cell vertices. The spatial distribution of cortical clues leads to the force field that determine final spindle apparatus orientation and the subsequent orientation of cell division.

Potassium channel

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Potassium_channel

Potassium channel Kv1.2, structure in a membrane-like environment. Calculated hydrocarbon boundaries of the lipid bilayer are indicated by red and blue lines.

Potassium channels are the most widely distributed type of ion channel found in virtually all organisms. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cell types and control a wide variety of cell functions.

Function

Potassium channels function to conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K⁺ ions in bulk water) and selectively (excluding, most notably, sodium despite the sub-angstrom difference in ionic radius). Biologically, these channels act to set or reset the resting potential in many cells. In excitable cells, such as neurons, the delayed counterflow of potassium ions shapes the action potential.

By contributing to the regulation of the cardiac action potential duration in cardiac muscle, malfunction of potassium channels may cause life-threatening arrhythmias. Potassium channels may also be involved in maintaining vascular tone.

They also regulate cellular processes such as the secretion of hormones (e.g., insulin release from beta-cells in the pancreas) so their malfunction can lead to diseases (such as diabetes).

Some toxins, such as dendrotoxin, are potent because they block potassium channels.

Types

There are four major classes of potassium channels:

• Calcium-activated potassium channel - open in response to the presence of calcium ions or other signalling molecules.
• Inwardly rectifying potassium channel - passes current (positive charge) more easily in the inward direction (into the cell).
• Tandem pore domain potassium channel - are constitutively open or possess high basal activation, such as the "resting potassium channels" or "leak channels" that set the negative membrane potential of neurons.
• Voltage-gated potassium channel - are voltage-gated ion channels that open or close in response to changes in the transmembrane voltage.

The following table contains a comparison of the major classes of potassium channels with representative examples (for a complete list of channels within each class, see the respective class pages).

For more examples of pharmacological modulators of potassium channels, see potassium channel blocker and potassium channel opener.

Potassium channel classes, function, and pharmacology.

Class	Subclasses	Function	Blockers	Activators
Calcium-activated 6T & 1P	BK channel SK channel IK channel	inhibition in response to rising intracellular calcium	Charybdotoxin, Iberiotoxin Apamin Some types of dendrotoxin from mamba snake venom, BKCa-specific GAL-021 Ethanol (alcohol)	1-EBIO NS309 CyPPA BKCa-specific: Flufenamic acid Meclofenamic acid Niflumic acid Nimesulide Rottlerin (mallotoxin) Tolfenamic acid
Inwardly rectifying 2T & 1P	ROMK (Kir1.1)	recycling and secretion of potassium in nephrons	Nonselective: Ba2+, Cs+
	Voltage-gated (Kir2.x)	final repolarization phase and stabilising the resting potential of the action potential in cardiac myocytes	Chloroquine Pentamidine ML133 Fluoxetine Nortryptiline Dronedarone Quinidine	Flecainide
	GPCR regulated (Kir3.x)	mediate the inhibitory effect of many GPCRs	Ifenprodil Cloperastine Tertiapin Tipepidine Ba2+	ML-297 (VU0456810)
	ATP-sensitive (Kir6.x)	close when ATP is high to promote insulin secretion	Acetohexamide  Carbutamide  Chlorpropamide  Glibenclamide (glyburide) Glibornuride  Glicaramide Gliclazide Glimepiride Glipizide	Bimakalim Cromakalim Diazoxide Levcromakalim Minoxidil Nicorandil Pinacidil
Tandem pore domain 4T & 2P	TWIK (TWIK-1, TWIK-2, KCNK7) TREK (TREK-1, TREK-2, TRAAK) TASK (TASK-1, TASK-3, TASK-5) TALK (TASK-2, TALK-1, TALK-2) THIK (THIK-1, THIK-2) TRESK	Contribute to resting potential	bupivacaine quinidine 12-O-Tetradecanoylphorbol-13-acetate Arachidonic acid Fluoxetine Norfluoxetine Modulated by GPCR agonists and antagonists, No selective blockers,	[citation needed] halothane Riluzole Volatile anaesthetics, e.g. Isoflurane Modulated by GPCR agonists and antagonists,
Voltage-gated 6T & 1P	hERG (Kv11.1) KvLQT1 (Kv7.1)	action potential repolarization limits frequency of action potentials (disturbances cause dysrhythmia)	3,4-Diaminopyridine (amifampridine) 4-Aminopyridine (fampridine/dalfampridine) Bretylium Tetraethylammonium hERG (KCNH2, Kv11.1)-specific: Ajmaline Amiodarone AmmTX3 KCNQ (Kv7)-specific: Linopirdine Ssm spooky toxin	KCNQ (Kv7)-specific: Flupirtine Retigabine (Kv7)

Structure

Top view of a potassium channel with potassium ions (purple) moving through the pore (in the center). (PDB: 1BL8​)

Potassium channels have a tetrameric structure in which four identical protein subunits associate to form a fourfold symmetric (C₄) complex arranged around a central ion conducting pore (i.e., a homotetramer). Alternatively four related but not identical protein subunits may associate to form heterotetrameric complexes with pseudo C₄ symmetry. All potassium channel subunits have a distinctive pore-loop structure that lines the top of the pore and is responsible for potassium selective permeability.

There are over 80 mammalian genes that encode potassium channel subunits. However potassium channels found in bacteria are amongst the most studied of ion channels, in terms of their molecular structure. Using X-ray crystallography,^[55][56] profound insights have been gained into how potassium ions pass through these channels and why (smaller) sodium ions do not.^[57] The 2003 Nobel Prize for Chemistry was awarded to Rod MacKinnon for his pioneering work in this area.^[58]

Selectivity filter

Crystallographic structure of the bacterial KcsA potassium channel (PDB: 1K4C​). In this figure, only two of the four subunits of the tetramer are displayed for the sake of clarity. The protein is displayed as a green cartoon diagram. In addition backbone carbonyl groups and threonine sidechain protein atoms (oxygen = red, carbon = green) are displayed. Finally potassium ions (occupying the S2 and S4 sites) and the oxygen atoms of water molecules (S1 and S3) are depicted as purple and red spheres respectively.

Potassium ion channels remove the hydration shell from the ion when it enters the selectivity filter. The selectivity filter is formed by a five residue sequence, TVGYG, termed the signature sequence, within each of the four subunits. This signature sequence is within a loop between the pore helix and TM2/6, historically termed the P-loop. This signature sequence is highly conserved, with the exception that a valine residue in prokaryotic potassium channels is often substituted with an isoleucine residue in eukaryotic channels. This sequence adopts a unique main chain structure, structurally analogous to a nest protein structural motif. The four sets of electronegative carbonyl oxygen atoms are aligned toward the center of the filter pore and form a square antiprism similar to a water-solvating shell around each potassium binding site. The distance between the carbonyl oxygens and potassium ions in the binding sites of the selectivity filter is the same as between water oxygens in the first hydration shell and a potassium ion in water solution, providing an energetically-favorable route for de-solvation of the ions. Sodium ions, however, are too small to fill the space between the carbonyl oxygen atoms. Thus, it is energetically favorable for sodium ions to remain bound with water molecules in the extracellular space, rather than to pass through the potassium-selective ion pore. This width appears to be maintained by hydrogen bonding and van der Waals forces within a sheet of aromatic amino acid residues surrounding the selectivity filter. The selectivity filter opens towards the extracellular solution, exposing four carbonyl oxygens in a glycine residue (Gly79 in KcsA). The next residue toward the extracellular side of the protein is the negatively charged Asp80 (KcsA). This residue together with the five filter residues form the pore that connects the water-filled cavity in the center of the protein with the extracellular solution.

Selectivity mechanism

The mechanism of potassium channel selectivity remains under continued debate. The carbonyl oxygens are strongly electro-negative and cation-attractive. The filter can accommodate potassium ions at 4 sites usually labelled S1 to S4 starting at the extracellular side. In addition, one ion can bind in the cavity at a site called SC or one or more ions at the extracellular side at more or less well-defined sites called S0 or Sext. Several different occupancies of these sites are possible. Since the X-ray structures are averages over many molecules, it is, however, not possible to deduce the actual occupancies directly from such a structure. In general, there is some disadvantage due to electrostatic repulsion to have two neighboring sites occupied by ions. Proposals for the mechanism of selectivity have been made based on molecular dynamics simulations, toy models of ion binding, thermodynamic calculations, topological considerations, and structural differences between selective and non-selective channels.

The mechanism for ion translocation in KcsA has been studied extensively by theoretical calculations and simulation. The prediction of an ion conduction mechanism in which the two doubly occupied states (S1, S3) and (S2, S4) play an essential role has been affirmed by both techniques. Molecular dynamics (MD) simulations suggest the two extracellular states, S_ext and S₀, reflecting ions entering and leaving the filter, also are important actors in ion conduction.

Hydrophobic region

This region neutralizes the environment around the potassium ion so that it is not attracted to any charges. In turn, it speeds up the reaction.

Central cavity

A central pore, 10 Å wide, is located near the center of the transmembrane channel, where the energy barrier is highest for the transversing ion due to the hydrophobity of the channel wall. The water-filled cavity and the polar C-terminus of the pore helices ease the energetic barrier for the ion. Repulsion by preceding multiple potassium ions is thought to aid the throughput of the ions. The presence of the cavity can be understood intuitively as one of the channel's mechanisms for overcoming the dielectric barrier, or repulsion by the low-dielectric membrane, by keeping the K⁺ ion in a watery, high-dielectric environment.

Regulation

Graphical representation of open and shut potassium channels (PDB: 1lnq​ and PDB: 1k4c​). Two simple bacterial channels are shown to compare the "open" channel structure on the right with the "closed" structure on the left. At top is the filter (selects potassium ions), and at bottom is the gating domain (controls opening and closing of channel).

The flux of ions through the potassium channel pore is regulated by two related processes, termed gating and inactivation. Gating is the opening or closing of the channel in response to stimuli, while inactivation is the rapid cessation of current from an open potassium channel and the suppression of the channel's ability to resume conducting. While both processes serve to regulate channel conductance, each process may be mediated by a number of mechanisms.

Generally, gating is thought to be mediated by additional structural domains which sense stimuli and in turn open the channel pore. These domains include the RCK domains of BK channels, and voltage sensor domains of voltage gated K⁺ channels. These domains are thought to respond to the stimuli by physically opening the intracellular gate of the pore domain, thereby allowing potassium ions to traverse the membrane. Some channels have multiple regulatory domains or accessory proteins, which can act to modulate the response to stimulus. While the mechanisms continue to be debated, there are known structures of a number of these regulatory domains, including RCK domains of prokaryotic and eukaryotic channels, pH gating domain of KcsA, cyclic nucleotide gating domains, and voltage gated potassium channels.

N-type inactivation is typically the faster inactivation mechanism, and is termed the "ball and chain" model. N-type inactivation involves interaction of the N-terminus of the channel, or an associated protein, which interacts with the pore domain and occludes the ion conduction pathway like a "ball". Alternatively, C-type inactivation is thought to occur within the selectivity filter itself, where structural changes within the filter render it non-conductive. There are a number of structural models of C-type inactivated K⁺ channel filters, although the precise mechanism remains unclear.

Pharmacology

Blockers

Main article: Potassium channel blocker

Potassium channel blockers inhibit the flow of potassium ions through the channel. They either compete with potassium binding within the selectivity filter or bind outside the filter to occlude ion conduction. An example of one of these competitors is quaternary ammonium ions, which bind at the extracellular face or central cavity of the channel. For blocking from the central cavity quaternary ammonium ions are also known as open channel blockers, as binding classically requires the prior opening of the cytoplasmic gate.

Barium ions can also block potassium channel currents, by binding with high affinity within the selectivity filter. This tight binding is thought to underlie barium toxicity by inhibiting potassium channel activity in excitable cells.

Medically potassium channel blockers, such as 4-aminopyridine and 3,4-diaminopyridine, have been investigated for the treatment of conditions such as multiple sclerosis. Off target drug effects can lead to drug induced Long QT syndrome, a potentially life-threatening condition. This is most frequently due to action on the hERG potassium channel in the heart. Accordingly, all new drugs are preclinically tested for cardiac safety.

Activators

Main article: Potassium channel opener

Muscarinic potassium channel

Birth of an Idea (2007) by Julian Voss-Andreae. The sculpture was commissioned by Roderick MacKinnon based on the molecule's atomic coordinates that were determined by MacKinnon's group in 2001.

See also: G protein-coupled inwardly-rectifying potassium channel

Some types of potassium channels are activated by muscarinic receptors and these are called muscarinic potassium channels (I_KACh). These channels are a heterotetramer composed of two GIRK1 and two GIRK4 subunits. Examples are potassium channels in the heart, which, when activated by parasympathetic signals through M2 muscarinic receptors, cause an outward current of potassium, which slows down the heart rate.

In fine art

Roderick MacKinnon commissioned Birth of an Idea, a 5-foot (1.5 m) tall sculpture based on the KcsA potassium channel. The artwork contains a wire object representing the channel's interior with a blown glass object representing the main cavity of the channel structure.

Amyloid

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Amyloid

Micrograph showing amyloid deposits (pink) in small bowel. Duodenum with amyloid deposition in lamina propria. Amyloid shows up as homogeneous pink material in lamina propria and around blood vessels. 20× magnification.

Amyloids are aggregates of proteins characterised by a fibrillar morphology of typically 7–13 nm in diameter, a β-sheet secondary structure (known as cross-β) and ability to be stained by particular dyes, such as Congo red. In the human body, amyloids have been linked to the development of various diseases. Pathogenic amyloids form when previously healthy proteins lose their normal structure and physiological functions (misfolding) and form fibrous deposits within and around cells. These protein misfolding and deposition processes disrupt the healthy function of tissues and organs.

Such amyloids have been associated with (but not necessarily as the cause of) more than 50 human diseases, known as amyloidosis, and may play a role in some neurodegenerative diseases. Some of these diseases are mainly sporadic and only a few cases are familial. Others are only familial. Some result from medical treatment. Prions are an infectious form of amyloids that can act as a template to convert other non-infectious forms. Amyloids may also have normal biological functions; for example, in the formation of fimbriae in some genera of bacteria, transmission of epigenetic traits in fungi, as well as pigment deposition and hormone release in humans.

Amyloids have been known to arise from many different proteins. These polypeptide chains generally form β-sheet structures that aggregate into long fibers; however, identical polypeptides can fold into multiple distinct amyloid conformations. The diversity of the conformations may have led to different forms of the prion diseases.

An unusual secondary structure named α sheet has been proposed as the toxic constituent of amyloid precursor proteins, but this idea is not widely accepted at present.

Amyloid of HET-s(218-289) prion pentamer, Podospora anserina (PDB: 2rnm​)

Definition

The name amyloid comes from the early mistaken identification by Rudolf Virchow of the substance as starch (amylum in Latin, from Ancient Greek: ἄμυλον, romanized: amylon), based on crude iodine-staining techniques. For a period, the scientific community debated whether or not amyloid deposits are fatty deposits or carbohydrate deposits until it was finally found (in 1859) that they are, in fact, deposits of albumoid proteinaceous material.

• The classical, histopathological definition of amyloid is an extracellular, proteinaceous fibrillar deposit exhibiting β-sheet secondary structure and identified by apple-green birefringence when stained with congo red under polarized light. These deposits often recruit various sugars and other components such as serum amyloid P component, resulting in complex, and sometimes inhomogeneous structures. Recently this definition has come into question as some classic, amyloid species have been observed in distinctly intracellular locations.
• A more recent, biophysical definition is broader, including any polypeptide that polymerizes to form a cross-β structure, in vivo or in vitro, inside or outside cells. Microbiologists, biochemists, biophysicists, chemists and physicists have largely adopted this definition, leading to some conflict in the biological community over an issue of language.

Proteins forming amyloids in diseases

To date, 37 human proteins have been found to form amyloid in pathology and be associated with well-defined diseases. The International Society of Amyloidosis classifies amyloid fibrils and their associated diseases based upon associated proteins (for example ATTR is the group of diseases and associated fibrils formed by TTR). A table is included below.

Protein	Diseases	Official abbreviation
β amyloid peptide (Aβ) from Amyloid precursor protein	Alzheimer's disease, Hereditary cerebral haemorrhage with amyloidosis	Aβ
α-synuclein	Parkinson's disease, Parkinson's disease dementia, Dementia with Lewy bodies, Multiple system atrophy	AαSyn
PrPSc	Transmissible spongiform encephalopathy (e.g. Fatal familial insomnia, Gerstmann-Sträussler-Scheinker disease, Creutzfeldt–Jakob disease, New variant Creutzfeldt–Jakob disease)	APrP
Microtubule-associated protein tau	Various forms of tauopathies (e.g. Pick's disease, Progressive supranuclear palsy, Corticobasal degeneration, Frontotemporal dementia with parkinsonism linked to chromosome 17, Argyrophilic grain disease)	ATau
Huntingtin exon 1	Huntington's disease	HTTex1
ABri peptide	Familial British dementia	ABri
ADan peptide	Familial Danish dementia	ADan
Fragments of immunoglobulin light chains	Light chain amyloidosis	AL
Fragments of immunoglobulin heavy chains	Heavy chain amyloidosis	AH
full length of N-terminal fragments of Serum amyloid A protein	AA amyloidosis	AA
Transthyretin	Senile systemic amyloidosis, Familial amyloid polyneuropathy, Familial amyloid cardiomyopathy, Leptomeningeal amyloidosis	ATTR
β-2 microglobulin	Dialysis related amyloidosis, Hereditary visceral amyloidosis (familial)	Aβ2M
N-terminal fragments of Apolipoprotein AI	ApoAI amyloidosis	AApoAI
C-terminally extended Apolipoprotein AII	ApoAII amyloidosis	AApoAII
N-terminal fragments of Apolipoprotein AIV	ApoAIV amyloidosis	AApoAIV
Apolipoprotein C-II	ApoCII amyloidosis	AApoCII
Apolipoprotein C-III	ApoCIII amyloidosis	AApoCIII
fragments of Gelsolin	Familial amyloidosis, Finnish type	AGel
Lysozyme	Hereditary non-neuropathic systemic amyloidosis	ALys
fragments of Fibrinogen α chain	Fibrinogen amyloidosis	AFib
N-terminally truncated Cystatin C	Hereditary cerebral hemorrhage with amyloidosis, Icelandic type	ACys
IAPP (Amylin)	Diabetes mellitus type 2, Insulinoma	AIAPP
Calcitonin	Medullary carcinoma of the thyroid	ACal
Atrial natriuretic factor	Cardiac arrhythmias, Isolated atrial amyloidosis	AANF
Prolactin	Pituitary prolactinoma	APro
Insulin	Injection-localized amyloidosis	AIns
Lactadherin / Medin	Aortic medial amyloidosis	AMed
Lactotransferrin / Lactoferrin	Gelatinous drop-like corneal dystrophy	ALac
Odontogenic ameloblast-associated protein	Calcifying epithelial odontogenic tumors	AOAAP
Pulmonary surfactant-associated protein C (SP-C)	Pulmonary alveolar proteinosis	ASPC
Leukocyte cell-derived chemotaxin-2 (LECT-2)	Renal LECT2 amyloidosis	ALECT2
Galectin-7	Lichen amyloidosis, Macular amyloidosis	AGal7
Corneodesmosin	Hypotrichosis simplex of the scalp	ACor
C-terminal fragments of TGFBI/Keratoepithelin	Lattice corneal dystrophy type I, Lattice corneal dystrophy type 3A, Lattice corneal dystrophy Avellino type	AKer
Semenogelin-1 (SGI)	Seminal vesicle amyloidosis	ASem1
Proteins S100A8/A9	Prostate cancer	none
Enfuvirtide	Injection-localized amyloidosis	AEnf

Non-disease and functional amyloids

Many examples of non-pathological amyloid with a well-defined physiological role have been identified in various organisms, including human. These may be termed as functional or physiological or native amyloid.

• Functional amyloid in Homo sapiens:
  • Intralumenal domain of melanocyte protein PMEL
  • Peptide/protein hormones stored as amyloids within endocrine secretory granules
  • Receptor-interacting serine/threonine-protein kinase 1/3 (RIP1/RIP3)
  • Fragments of prostatic acid phosphatase and semenogelins
• Functional amyloid in other organisms:
  • Curli fibrils produced by E. coli, Salmonella, and a few other members of the Enterobacteriales (Csg). The genetic elements (operons) encoding the curli system are phylogenetic widespread and can be found in at least four bacterial phyla. This suggest that many more bacteria may express curli fibrils.
  • GvpA, forming the walls of particular Gas vesicles, i.e. the buoyancy organelles of aquatic archaea and eubacteria
  • Fap fibrils in various species of Pseudomonas
  • Chaplins from Streptomyces coelicolor
  • Spidroin from Trichonephila edulis (spider) (Spider silk)
  • Hydrophobins from Neurospora crassa and other fungi
  • Fungal cell adhesion proteins forming cell surface amyloid regions with greatly increased binding strength
  • Environmental biofilms according to staining with amyloid specific dyes and antibodies.
  • Tubular sheaths encasing Methanosaeta thermophila filaments
• Functional amyloid acting as prions
  • Several yeast prions are based on an infectious amyloid, e.g. [PSI+] (Sup35p); [URE3] (Ure2p); [PIN+] or [RNQ+] (Rnq1p); [SWI1+] (Swi1p) and [OCT8+] (Cyc8p)
  • Prion HET-s from Podospora anserina
  • Neuron-specific isoform of CPEB from Aplysia californica (marine snail)

Structure

Structure of a fibril, consisting of one single protofilament, of the amyloid β peptide viewed down the long axis of the fibril (PDB: 2mlq​)

Amyloids are formed of long unbranched fibers that are characterized by an extended β-sheet secondary structure in which individual β strands (β-strands) (coloured arrows in the adjacent figure) are arranged in an orientation perpendicular to the long axis of the fiber. Such a structure is known as cross-β structure. Each individual fiber may be 7–13 nanometres in width and a few micrometres in length. The main hallmarks recognised by different disciplines to classify protein aggregates as amyloid is the presence of a fibrillar morphology with the expected diameter, detected using transmission electron microscopy (TEM) or atomic force microscopy (AFM), the presence of a cross-β secondary structure, determined with circular dichroism, FTIR, solid-state nuclear magnetic resonance (ssNMR), X-ray crystallography, or X-ray fiber diffraction (often considered the "gold-standard" test to see whether a structure contains cross-β fibres), and an ability to stain with specific dyes, such as Congo red, thioflavin T or thioflavin S.

The term "cross-β" was based on the observation of two sets of diffraction lines, one longitudinal and one transverse, that form a characteristic "cross" pattern. There are two characteristic scattering diffraction signals produced at 4.7 and 10 Å (0.47 nm and 1.0 nm), corresponding to the interstrand and stacking distances in β sheets. The "stacks" of β sheet are short and traverse the breadth of the amyloid fibril; the length of the amyloid fibril is built by aligned β-strands. The cross-β pattern is considered a diagnostic hallmark of amyloid structure.

Amyloid fibrils are generally composed of 1–8 protofilaments (one protofilament also corresponding to a fibril is shown in the figure), each 2–7 nm in diameter, that interact laterally as flat ribbons that maintain the height of 2–7 nm (that of a single protofilament) and are up to 30 nm wide; more often protofilaments twist around each other to form the typically 7–13 nm wide fibrils. Each protofilament possesses the typical cross-β structure and may be formed by 1–6 β-sheets (six are shown in the figure) stacked on each other. Each individual protein molecule can contribute one to several β-strands in each protofilament and the strands can be arranged in antiparallel β-sheets, but more often in parallel β-sheets. Only a fraction of the polypeptide chain is in a β-strand conformation in the fibrils, the remainder forms structured or unstructured loops or tails.

For a long time our knowledge of the atomic-level structure of amyloid fibrils was limited by the fact that they are unsuitable for the most traditional methods for studying protein structures. Recent years have seen progress in experimental methods, including solid-state NMR spectroscopy and Cryo-Electron Microscopy. Combined, these methods have provided 3D atomic structures of amyloid fibrils formed by amyloid β peptides, α-synuclein, tau, and the FUS protein, associated with various neurodegenerative diseases.

X-ray diffraction studies of microcrystals revealed atomistic details of core region of amyloid, although only for simplified peptides having a length remarkably shorter than that of peptides or proteins involved in disease. The crystallographic structures show that short stretches from amyloid-prone regions of amyloidogenic proteins run perpendicular to the filament axis, consistent with the "cross-β" feature of amyloid structure. They also reveal a number of characteristics of amyloid structures – neighboring β-sheets are tightly packed together via an interface devoid of water (therefore referred to as dry interface), with the opposing β-strands slightly offset from each other such that their side-chains interdigitate. This compact dehydrated interface created was termed a steric-zipper interface. There are eight theoretical classes of steric-zipper interfaces, dictated by the directionality of the β-sheets (parallel and anti-parallel) and symmetry between adjacent β-sheets. A limitation of X-ray crystallography for solving amyloid structure is represented by the need to form microcrystals, which can be achieved only with peptides shorter than those associated with disease.

Although bona fide amyloid structures always are based on intermolecular β-sheets, different types of "higher order" tertiary folds have been observed or proposed. The β-sheets may form a β-sandwich, or a β-solenoid which may be either β-helix or β-roll. Native-like amyloid fibrils in which native β-sheet containing proteins maintain their native-like structure in the fibrils have also been proposed. There are few developed ideas on how the complex backbone topologies of disulfide-constrained proteins, which are prone to form amyloid fibrils (such as insulin and lysozyme), adopt the amyloid β-sheet motif. The presence of multiple constraints significantly reduces the accessible conformational space, making computational simulations of amyloid structures more feasible. 

One complicating factor in studies of amyloidogenic polypeptides is that identical polypeptides can fold into multiple distinct amyloid conformations. This phenomenon is typically described as amyloid polymorphism. It has notable biological consequences given that it is thought to explain the prion strain phenomenon.

Formation

Three phases of amyloid fibril formation: lag phase, exponential phase and plateau phase

Amyloid is formed through the polymerization of hundreds to thousands of monomeric peptides or proteins into long fibers. Amyloid formation involves a lag phase (also called nucleation phase), an exponential phase (also called growth phase) and a plateau phase (also called saturation phase), as shown in the figure. Indeed, when the quantity of fibrils is plotted versus time, a sigmoidal time course is observed reflecting the three distinct phases.

In the simplest model of 'nucleated polymerization' (marked by red arrows in the figure below), individual unfolded or partially unfolded polypeptide chains (monomers) convert into a nucleus (monomer or oligomer) via a thermodynamically unfavourable process that occurs early in the lag phase. Fibrils grow subsequently from these nuclei through the addition of monomers in the exponential phase.

A different model, called 'nucleated conformational conversion' and marked by blue arrows in the figure below, was introduced later on to fit some experimental observations: monomers have often been found to convert rapidly into misfolded and highly disorganized oligomers distinct from nuclei. Only later on, will these aggregates reorganise structurally into nuclei, on which other disorganised oligomers will add and reorganise through a templating or induced-fit mechanism (this 'nucleated conformational conversion' model), eventually forming fibrils.

Normally folded proteins have to unfold partially before aggregation can take place through one of these mechanisms. In some cases, however, folded proteins can aggregate without crossing the major energy barrier for unfolding, by populating native-like conformations as a consequence of thermal fluctuations, ligand release or local unfolding occurring in particular circumstances. In these native-like conformations, segments that are normally buried or structured in the fully folded and possessing a high propensity to aggregate become exposed to the solvent or flexible, allowing the formation of native-like aggregates, which convert subsequently into nuclei and fibrils. This process is called 'native-like aggregation' (green arrows in the figure) and is similar to the 'nucleated conformational conversion' model.

A more recent, modern and thorough model of amyloid fibril formation involves the intervention of secondary events, such as 'fragmentation', in which a fibril breaks into two or more shorter fibrils, and 'secondary nucleation', in which fibril surfaces (not fibril ends) catalyze the formation of new nuclei. Both secondary events increase the number of fibril ends able to recruit new monomers or oligomers, therefore accelerating fibril formation through a positive feedback mechanism. These events add to the well recognised steps of primary nucleation (formation of the nucleus from the monomers through one of models described above), fibril elongation (addition of monomers or oligomers to growing fibril ends) and dissociation (opposite process).

Such a new model is described in the figure on the right and involves the utilization of a master equation that includes all steps of amyloid fibril formation, i.e. primary nucleation, fibril elongation, secondary nucleation and fibril fragmentation. The rate constants of the various steps can be determined from a global fit of a number of time courses of aggregation (for example ThT fluorescence emission versus time) recorded at different protein concentrations. The general master equation approach to amyloid fibril formation with secondary pathways has been developed by Knowles, Vendruscolo, Cohen, Michaels and coworkers and considers the time evolution of the concentration ${\displaystyle f(t,j)}$ of fibrils of length ${\displaystyle j}$ (here ${\displaystyle j}$ represents the number of monomers in an aggregate). $${\displaystyle \begin{array}{rl}\frac{\mathrm{\partial }f(t,j)}{\mathrm{\partial }t}& =2k_{+}m(t)f(t,j-1)-2k_{+}m(t)f(t,j)\\ & +2k_{\mathrm{o}\mathrm{f}\mathrm{f}}f(t,j+1)-2k_{\mathrm{o}\mathrm{f}\mathrm{f}}f(t,j)\\ & +k_{-}\sum _{i=j+1}^{\mathrm{\infty }}f(t,i)-k_{-}(j-1)f(t,j)\\ & +k_{1}m(t{)}^{n_1}{\delta }_{j,n_1}+k_{2}m(t{)}^{n_2}M(t){\delta }_{j,n_2}\\ \end{array}}$$where ${\displaystyle {\delta }_{i,j}}$ denotes the Kronecker delta. The physical interpretation of the various terms in the above master equation is straight forward: the terms on the first line describe the growth of fibrils via monomer addition with rate constant ${\displaystyle k_{+}}$ (elongation). The terms on the second line describe monomer dissociation, i.e. the inverse process of elongation. ${\displaystyle k_{\mathrm{o}\mathrm{f}\mathrm{f}}}$ is the rate constant of monomer dissociation. The terms on the third line describe the effect of fragmentation, which is assumed to occur homogeneously along fibrils with rate constant ${\displaystyle k_{-}}$. Finally, the terms on the last line describe primary and secondary nucleation respectively. Note that the rate of secondary nucleation is proportional to the mass of aggregates, defined as ${\displaystyle M(t)=\sum _{j=n_1}^{\mathrm{\infty }}jf(t,j)}$.

Following this analytical approach, it has become apparent that the lag phase does not correspond necessarily to only nucleus formation, but rather results from a combination of various steps. Similarly, the exponential phase is not only fibril elongation, but results from a combination of various steps, involving primary nucleation, fibril elongation, but also secondary events. A significant quantity of fibrils resulting from primary nucleation and fibril elongation may be formed during the lag phase and secondary steps, rather than only fibril elongation, can be the dominant processes contributing to fibril growth during the exponential phase. With this new model, any perturbing agents of amyloid fibril formation, such as putative drugs, metabolites, mutations, chaperones, etc., can be assigned to a specific step of fibril formation.

Amino acid sequence and amyloid formation

In general, amyloid polymerization (aggregation or non-covalent polymerization) is sequence-sensitive, that is mutations in the sequence can induce or prevent self-assembly. For example, humans produce amylin, an amyloidogenic peptide associated with type II diabetes, but in rats and mice prolines are substituted in critical locations and amyloidogenesis does not occur. Studies comparing synthetic to recombinant β amyloid peptide in assays measuring rate of fibrillation, fibril homogeneity, and cellular toxicity showed that recombinant β amyloid peptide has a faster fibrillation rate and greater toxicity than synthetic β amyloid peptide.

There are multiple classes of amyloid-forming polypeptide sequences. Glutamine-rich polypeptides are important in the amyloidogenesis of Yeast and mammalian prions, as well as trinucleotide repeat disorders including Huntington's disease. When glutamine-rich polypeptides are in a β-sheet conformation, glutamines can brace the structure by forming inter-strand hydrogen bonding between its amide carbonyls and nitrogens of both the backbone and side chains. The onset age for Huntington's disease shows an inverse correlation with the length of the polyglutamine sequence, with analogous findings in a C. elegans model system with engineered polyglutamine peptides.

Other polypeptides and proteins such as amylin and the β amyloid peptide do not have a simple consensus sequence and are thought to aggregate through the sequence segments enriched with hydrophobic residues, or residues with high propensity to form β-sheet structure. Among the hydrophobic residues, aromatic amino-acids are found to have the highest amyloidogenic propensity.

Cross-polymerization (fibrils of one polypeptide sequence causing other fibrils of another sequence to form) is observed in vitro and possibly in vivo. This phenomenon is important, since it would explain interspecies prion propagation and differential rates of prion propagation, as well as a statistical link between Alzheimer's and type 2 diabetes. In general, the more similar the peptide sequence the more efficient cross-polymerization is, though entirely dissimilar sequences can cross-polymerize and highly similar sequences can even be "blockers" that prevent polymerization.

Amyloid toxicity

The reasons why amyloid cause diseases are unclear. In some cases, the deposits physically disrupt tissue architecture, suggesting disruption of function by some bulk process. An emerging consensus implicates prefibrillar intermediates, rather than mature amyloid fibers, in causing cell death, particularly in neurodegenerative diseases.^[17][69] The fibrils are, however, far from innocuous, as they keep the protein homeostasis network engaged, release oligomers, cause the formation of toxic oligomers via secondary nucleation, grow indefinitely spreading from district to district^[2] and, in some cases, may be toxic themselves.^[70]

Calcium dysregulation has been observed to occur early in cells exposed to protein oligomers. These small aggregates can form ion channels through lipid bilayer membranes and activate NMDA and AMPA receptors. Channel formation has been hypothesized to account for calcium dysregulation and mitochondrial dysfunction by allowing indiscriminate leakage of ions across cell membranes.^[71] Studies have shown that amyloid deposition is associated with mitochondrial dysfunction and a resulting generation of reactive oxygen species (ROS), which can initiate a signalling pathway leading to apoptosis.^[72] There are reports that indicate amyloid polymers (such as those of huntingtin, associated with Huntington's disease) can induce the polymerization of essential amyloidogenic proteins, which should be deleterious to cells. Also, interaction partners of these essential proteins can also be sequestered.^[73]

All these mechanisms of toxicity are likely to play a role. In fact, the aggregation of a protein generates a variety of aggregates, all of which are likely to be toxic to some degree. A wide variety of biochemical, physiological and cytological perturbations has been identified following the exposure of cells and animals to such species, independently of their identity. The oligomers have also been reported to interact with a variety of molecular targets. Hence, it is unlikely that there is a unique mechanism of toxicity or a unique cascade of cellular events. The misfolded nature of protein aggregates causes a multitude of aberrant interactions with a multitude of cellular components, including membranes, protein receptors, soluble proteins, RNAs, small metabolites, etc.

Histological staining

In the clinical setting, amyloid diseases are typically identified by a change in the spectroscopic properties of planar aromatic dyes such as thioflavin T, congo red or NIAD-4. In general, this is attributed to the environmental change, as these dyes intercalate between β-strands to confine their structure.

Congo Red positivity remains the gold standard for diagnosis of amyloidosis. In general, binding of Congo Red to amyloid plaques produces a typical apple-green birefringence when viewed under cross-polarized light. Recently, significant enhancement of fluorescence quantum yield of NIAD-4 was exploited to super-resolution fluorescence imaging of amyloid fibrils and oligomers. To avoid nonspecific staining, other histology stains, such as the hematoxylin and eosin stain, are used to quench the dyes' activity in other places such as the nucleus, where the dye might bind. Modern antibody technology and immunohistochemistry has made specific staining easier, but often this can cause trouble because epitopes can be concealed in the amyloid fold; in general, an amyloid protein structure is a different conformation from the one that the antibody recognizes.