Components of the Cytoskeleton and their Important Functions in Reproductive Biology
Cytoskeletal functions are essential for proper oocyte maturation, fertilization, and mammalian development to accommodate an enormous diversity of cellular needs and enable development into a healthy embryo.
The cytoskeleton, comprising a complex network of fibers, primarily consists of three families of protein molecules that assemble to form three main types of filaments: microtubules, intermediate filaments, and microfilaments. In more recent years, the cytoskeletal network has been expanded to include septins and centrosomes (reviewed in Schatten and Sun, 2015b). Hundreds of accessory proteins link these cytoskeletal components to each other and to various cell organelles, vesicles, macromolecular assemblies, and specific molecules to facilitate intra- and intercellular communications and signal transductions for specific functions that are vitally important for embryo development. In the following, the major cytoskeletal components are briefly described. More specific information is available in previous reviews (Gallo and Tosti 2015; Li and Hu 2015; Schatten and Sun 2015b; Suzuki 2015 a,b) and in the specific sections of this chapter that follow.
Microtubules and tubulins: Microtubules are highly dynamic fibers composed of a/fi tubulin subunit heterodimers that are typically (but not always) assembled into laterally associated 13 protofilaments to compose one single cylindrical complete microtubule with an outer diameter of around 25 nm. Individual microtubules undergo dynamic phases of growth (polymerization) and shrinkage (depolymerization), which preferentially takes place at their plus (+) and their minus (-) ends, respectively. This biochemical process is generally referred to as “dynamic instability" that allows dynamic modulation of microtubules and their varied specific functions. Various mechanisms are in place to regulate microtubule stability. For example, the microtubule minus end can be stabilized by attachment to microtubule organizing centers (MTOCs; centrosomes), the Golgi apparatus, or cell membranes. Microtubule dynamics and functions can further be influenced by Ca++, pH, microtubule-associated proteins (MAPS), and posttranslational modifications (PTMs) such as acetylation and detyrosination/tyrosination which allows variability in dynamics, remodeling of the microtubule network, interactions with cellular components, and microtubule motor proteins such as dynein and kinesin. Microtubule motor proteins are critically important for directed transport along microtubules to serve specific functions including cellular metabolism and intracellular signal transduction (reviewed in Schatten and Sun, 2014).
Experimentally, microtubule drugs are frequently used to either inhibit microtubule polymerization (colcemid, colchicine, nocoda- zole, podophyllotoxin, and griseofulvin) or prevent depolymerization (taxol, paclitaxel), thereby inhibiting specific microtubule functions that require microtubule dynamics. Several of these drugs are being used for clinical applications and include treatment of cancer (reviewed in Schatten and Sun, 2012, 2015b). Microtubule dysfunctions are associated with diseases and disorders such as Alzheimer’s and Parkinson’s and they are further observed in aging cells, which is relevant for aging oocyte cells, as will be discussed in Section 10.4.
The tubulin family further includes у-tubulin that is well known for its microtubule nucleating functions (reviewed in Schatten 2008; Schatten and Sun 2011a,b; 2015a,b). It is mainly associated with microtubule nucleation at centrosomes but it is also localized at the Golgi and the plasma membrane where microtubule nucleation can take place. In non-polarized interphase cells the majority of microtubules are nucleated by у-tubulin from centrosomes but microtubule nucleation and organization changes in polarized cells when microtubules become organized by у-tubulin from the cell surface to aid in the cellular polarization process, as will be discussed in Section 10.6.
Four more tubulins are known, which are delta (Д)-, epsilon (e)-, zeta (C)-, and eta (^)-tubulins (McKean et al., 2001). Functions of these tubulins are mainly linked to eukaryotic centrioles and/or basal bodies (reviewed in Schatten and Sun 2015b).
Intermediate filaments: Intermediate filaments are the most stable of all cytoskeletal fibers. They are ropelike fibers with a diameter of ~10nm comprising a large and heterogeneous family of at least 65 different proteins subcategorized into six different types or classes in vertebrates (reviewed in Li et al., 2015; Suzuki 2015b; Wang 2015). Intermediate filaments play a role in cellular communication and intermediate filament dysfunctions have been implicated in communication disorders between cells as well as in laminopathies in which dysfunctions of lamins, a type of intermediate filaments comprising a meshwork of filaments underlining the inner nuclear envelope, occur.
Microfilaments: Microfilaments are composed of actin monomer molecules (g-actin; globular actin) to form filamentous (F) actin or microfilaments with a typical diameter of approximately 7-8nm. Microfilament functions include membrane trafficking, cell shape changes, and signaling functions including those in microvilli (reviewed in Brayford et al., 2015; McMichael and Lee, 2015; Shi- rao and Koganezawa, 2015). Numerous actin binding proteins are known that allow organization into linear bundles, two-dimensional networks, and three-dimensional gels. Microfilament functions are facilitated by microfilament-associated and microfilament-interacting molecules. The actin nucleating Arp2/3 (actin-related protein 2/3) complex is important for the formation of new actin filaments off the sides of existing microfilaments (reviewed by Sun and Kim 2013). Microfilament dynamics can be influenced experimentally by drugs that prevent polymerization (such as cytochalasin drugs) or that prevent depolymerization (such as phalloidin) (reviewed in Schatten and Sun 2015b).
Centrosomes: Centrosomes are major Microtubule Organizing Centers (MTOCs) that nucleate and organize microtubules during interphase and mitosis. These non-membrane-bound organelles are composed of numerous centrosomal proteins that are embedded in a proteinaceous matrix structure. Aside from serving as MTOC the centrosome also serves as major cellular communication center and orchestrates signal transduction processes through its microtubule network. In interphase, the centrosome is closely associated with the nucleus and organizes a radial microtubule aster. It duplicates during S/G2 and separates after nuclear envelope breakdown in early prophase; it becomes localized to the mitotic poles during mitosis (reviewed in Schatten 2008; Schatten and Sun 2012; 2015b). At least 60 centrosomal proteins are known for centrosomes in somatic interphase cells (reviewed in Wilkinson et al., 2004), which does not include centrosomal proteins that transiently associate with centrosomes for cell signaling and cell cycle-specific functions. As mentioned previously, у-tubulin is the major protein for microtubule nucleation. It is integrated in the large у-tubulin-ring complex (yTuRC) associated with the centrosome structure (reviewed in Schatten 2008; Schatten and Sun 2015a,b). Other well-studied centro- somal proteins include pericentrin, and centrin (reviewed in Schatten 2008) as well as the centrosome-associated protein NuMA (Sun and Schatten 2006; Liu et al., 2006; Alvarez-Sedo et al., 2011). As will be discussed later, a typical somatic cell centrosome contains a pair of centrioles (reviewed in Schatten 2008) while acentriolar centrosomes are typical for mammalian oocytes (reviewed in Schatten and Sun 2011a,b). The number of centrosomal proteins embedded in the proteinacous centrosome matrix vary greatly in different somatic and reproductive cells with specific differences in different animal species (reviewed in Schatten 2008; Schatten and Sun 2011a,b; 2015a,b). Detailed information on centrosomes is available in several previous reviews (Schatten 2008; Schatten and Sun 2011a,b; 2015a,b).
Septins: Septins represent a novel unconventional component of the cytoskeleton (Mostowy and Pascale 2012). They are a family of proteins that can form non-polar filaments or rings. Septins play a role in cytokinesis by recruiting different proteins to the contractile ring. They can interact with the actin and microtubule cytoskeleton.
In mouse oocytes, Septin2 is posttranslationally modified by SUMOy- lation and is required for chromosome congression (Zhu et al., 2010). Septin 1 is required for spindle assembly and chromosome congression (Zhu et al., 2011) while Septin 7 is required for meiosis (Li et al., 2012).
Specific aspects of these cytoskeletal components will be highlighted and discussed in the specific sections that follow with focus on cytoskeletal functions and dynamics in human oocyte maturation, fertilization, cell division, cellular differentiation and embryo development.
