Dynactin is a 23 subunit protein complex that acts as a co-factor for the microtubule motor cytoplasmic dynein-1. It is built around a short filament of actin related protein-1 (Arp1). [1] [2]
Dynactin is a 23 subunit protein complex that acts as a co-factor for the microtubule motor cytoplasmic dynein-1. It is built around a short filament of actin related protein-1 (Arp1). [1] [2]
Dynactin was identified as an activity that allowed purified cytoplasmic dynein to move membrane vesicles along microtubules in vitro. [3] It was shown to be a multiprotein complex and named "dynactin" because of its role in dynein activation. [4]
The main features of dynactin were visualized by quick-freeze, deep-etch, rotary shadow electron microscopy. It appears as a short filament, 37-nm in length, which resembles F-actin, plus a thinner, laterally oriented arm. [5] Antibody labelling was used to map the location of the dynactin subunits. [5] [6]
Dynactin consists of three major structural domains: (1) sidearm-shoulder: DCTN1/p150Glued, DCTN2/p50/dynamitin, DCTN3/p24/p22;(2)the Arp1 filament: ACTR1A/Arp1/centractin, actin, CapZ; and (3) the pointed end complex: Actr10/Arp11, DCTN4/p62, DCTN5/p25, and DCTN6/p27. [1]
A 4Å cryo-EM structure of dynactin [7] revealed that its filament contains eight Arp1 molecules, one β-actin and one Arp11. In the pointed end complex p62/DCTN4 binds to Arp11 and β-actin and p25 and p27 bind both p62 and Arp11. At the barbed end the capping protein (CapZαβ) binds the Arp1 filament in the same way that it binds actin, although with more charge complementarity, explaining why it binds dynactin more tightly than actin. [8]
The shoulder contains two copies of p150Glued/DCTN1, four copies of p50/DCTN2 and two copies of p24/DCTN3. [1] These proteins form long bundles of alpha helices, which wrap over each other and contact the Arp1 filament. [7] The N-termini of p50/DCTN2 emerge from the shoulder and coat the filament, providing a mechanism for controlling the filament length. [7] The C-termini of the p150Glued/DCTN1 dimer are embedded in the shoulder, whereas the N-terminal 1227 amino acids form the projecting arm. The arm consists of an N-terminal CAPGly domain which can bind the C-terminal tails of microtubules and the microtubule plus end binding protein EB1. This is followed by a basic region, also involved in microtubule binding, a folded-back coiled coil (CC1), the intercoiled domain (ICD) and a second coiled coil domain (CC2). [7] The p150Glued arm can dock into against the side of the Arp1 filament and pointed end complex. [7]
DCTN2 (dynamitin) is also involved in anchoring microtubules to centrosomes and may play a role in synapse formation during brain development. [9] Arp1 has been suggested as the domain for dynactin binding to membrane vesicles (such as Golgi or late endosome) through its association with β-spectrin. [10] [11] [12] [13] The pointed end complex (PEC) has been shown to be involved in selective cargo binding. PEC subunits p62/DCTN4 and Arp11/Actr10 are essential for dynactin complex integrity and dynactin/dynein targeting to the nuclear envelope before mitosis. [14] [15] [16] Actr10 along with Drp1 (Dynamin related protein 1) have been documented as vital to the attachment of mitochondria to the dynactin complex. [17] Dynactin p25/DCTN5 and p27/DCTN6 are not essential for dynactin complex integrity, but are required for early and recycling endosome transport during the interphase and regulation of the spindle assembly checkpoint in mitosis. [16] [18] [19]
Dynein and dynactin were reported to interact directly by the binding of dynein intermediate chains with p150Glued. [20] The affinity of this interaction is around 3.5μM. [21] Dynein and dynactin do not run together in a sucrose gradient, but can be induced to form a tight complex in the presence of the N-terminal 400 amino acids of Bicaudal D2 (BICD2), a cargo adaptor that links dynein and dynactin to Golgi derived vesicles. [22] In the presence of BICD2, dynactin binds to dynein and activates it to move for long distances along microtubules. [23] [24]
A cryo-EM structure of dynein, dynactin and BICD2 [7] showed that the BICD2 coiled coil runs along the dynactin filament. The tail of dynein also binds to the Arp1 filament, sitting in the equivalent site that myosin uses to bind actin. The contacts between the dynein tail and dynactin all involve BICD, explaining why it is needed to bring them together. The dynein/dynactin/BICD2 (DDB) complex has also been observed, by negative stain EM, on microtubules. This shows that the cargo (Rab6) binding end of BICD2 extends out through the pointed end complex at the opposite end away from the dynein motor domains. [25]
Dynactin is often essential for dynein activity [1] [3] and can be thought of as a "dynein receptor" [20] that modulates binding of dynein to cell organelles which are to be transported along microtubules. [26] [27] Dynactin also enhances the processivity of cytoplasmic dynein [28] and kinesin-2 motors. [29] Dynactin is involved in various processes like chromosome alignment and spindle organization [30] in cell division. [31] Dynactin contributes to mitotic spindle pole focusing through its binding to nuclear mitotic apparatus protein (NuMA). [32] [33] Dynactin also targets to the kinetochore through binding between DCTN2/dynamitin and zw10 and has a role in mitotic spindle checkpoint inactivation. [34] [35] During prometaphase, dynactin also helps target polo-like kinase 1 (Plk1) to kinetochores through cyclin dependent kinase 1 (Cdk1)-phosphorylated DCTN6/p27, which is involved in proper microtubule-kinetochore attachment and recruitment of spindle assembly checkpoint protein Mad1. [19] In addition, dynactin has been shown to play an essential role in maintaining nuclear position in Drosophila, [36] zebrafish [37] or in different fungi. [38] [39] Dynein and dynactin concentrate on the nuclear envelope during the prophase and facilitate nuclear envelope breakdown via its DCTN4/p62 and Arp11 subunits. [16] [14] Dynactin is also required for microtubule anchoring at centrosomes and centrosome integrity. [40] Destabilization of the centrosomal pool of dynactin also causes abnormal G1 centriole separation and delayed entry into S phase, suggesting that dynactin contributes to the recruitment of important cell cycle regulators to centrosomes. [41] In addition to transport of various organelles in the cytoplasm, dynactin also links kinesin II to organelles. [42]
Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nm and have an inner diameter between 11 and 15 nm. They are formed by the polymerization of a dimer of two globular proteins, alpha and beta tubulin into protofilaments that can then associate laterally to form a hollow tube, the microtubule. The most common form of a microtubule consists of 13 protofilaments in the tubular arrangement.
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.
Dyneins are a family of cytoskeletal motor proteins that move along microtubules in cells. They convert the chemical energy stored in ATP to mechanical work. Dynein transports various cellular cargos, provides forces and displacements important in mitosis, and drives the beat of eukaryotic cilia and flagella. All of these functions rely on dynein's ability to move towards the minus-end of the microtubules, known as retrograde transport; thus, they are called "minus-end directed motors". In contrast, most kinesin motor proteins move toward the microtubules' plus-end, in what is called anterograde transport.
A kinetochore is a disc-shaped protein structure associated with duplicated chromatids in eukaryotic cells where the spindle fibers attach during cell division to pull sister chromatids apart. The kinetochore assembles on the centromere and links the chromosome to microtubule polymers from the mitotic spindle during mitosis and meiosis. The term kinetochore was first used in a footnote in a 1934 Cytology book by Lester W. Sharp and commonly accepted in 1936. Sharp's footnote reads: "The convenient term kinetochore has been suggested to the author by J. A. Moore", likely referring to John Alexander Moore who had joined Columbia University as a freshman in 1932.
Motor proteins are a class of molecular motors that can move along the cytoplasm of cells. They convert chemical energy into mechanical work by the hydrolysis of ATP. Flagellar rotation, however, is powered by a proton pump.
In cell biology, microtubule nucleation is the event that initiates de novo formation of microtubules (MTs). These filaments of the cytoskeleton typically form through polymerization of α- and β-tubulin dimers, the basic building blocks of the microtubule, which initially interact to nucleate a seed from which the filament elongates.
Dynactin subunit 1 is a protein that in humans is encoded by the DCTN1 gene.
Tubulin alpha-4A chain is a protein that in humans is encoded by the TUBA4A gene.
Dynactin subunit 2 is a protein that in humans is encoded by the DCTN2 gene
Microtubule-associated protein RP/EB family member 1 is a protein that in humans is encoded by the MAPRE1 gene.
Alpha-centractin (yeast) or ARP1 is a protein that in humans is encoded by the ACTR1A gene.
Cytoplasmic dynein 1 heavy chain 1 is a protein that in humans is encoded by the DYNC1H1 gene.
Centromere/kinetochore protein zw10 homolog is a protein that in humans is encoded by the ZW10 gene. This gene encodes a protein that is one of many involved in mechanisms to ensure proper chromosome segregation during cell division. The encoded protein binds to centromeres during the prophase, metaphase, and early anaphase cell division stages and to kinetochore microtubules during metaphase.
Dynactin subunit 3 is a protein that in humans is encoded by the DCTN3 gene.
Beta-centractin is a protein that in humans is encoded by the ACTR1B gene.
Arp2/3 complex is a seven-subunit protein complex 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 regulation of rearrangements of the actin cytoskeleton is important for processes like cell locomotion, phagocytosis, and intracellular motility of lipid vesicles.
Dynactin 5 (p25) is a protein that in humans is encoded by the DCTN5 gene.
The LINC complex is a protein complex associated with both inner and outer membranes of the nucleus. It is composed of SUN-domain proteins and KASH-domain proteins. The SUN-domain proteins are associated with both nuclear lamins and chromatin and cross the inner nuclear membrane. They interact with the KASH domain proteins in the perinuclear (lumen) space between the two membranes. The KASH domain proteins cross the outer nuclear membrane and interact with actin filaments, microtubule filaments, intermediate filaments, centrosomes and cytoplasmic organelles. The number of SUN-domain and KASH-domain proteins increased in evolution.
In molecular biology, DCTN6 is that subunit of the dynactin protein complex that is encoded by the p27 gene. Dynactin is the essential component for microtubule-based cytoplasmic dynein motor activity in intracellular transport of a variety of cargoes and organelles.
Microtubule plus-end/positive-end tracking proteins or +TIPs are a type of microtubule associated protein (MAP) which accumulate at the plus ends of microtubules. +TIPs are arranged in diverse groups which are classified based on their structural components; however, all classifications are distinguished by their specific accumulation at the plus end of microtubules and their ability to maintain interactions between themselves and other +TIPs regardless of type. +TIPs can be either membrane bound or cytoplasmic, depending on the type of +TIPs. Most +TIPs track the ends of extending microtubules in a non-autonomous manner.