pericentrin-like protein: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Pericentrin-like protein

Synonyms - D-PLP

Cytological map position - 71B2--4

Function - scaffolding protein

Keywords - centriole, pericentriolar material, cilia and flagella, PNS

Symbol - Plp

FlyBase ID: FBgn0086690

Genetic map position - 3-

Classification - PACT domain protein

Cellular location - cytoplasmic/centriolar



NCBI links: | EntrezGene | | HomoloGene |
Recent literature
Lerit, D.A., Jordan, H.A., Poulton, J.S., Fagerstrom, C.J., Galletta, B.J., Peifer, M. and Rusan, N.M. (2015). Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function. J Cell Biol 210: 79-97. PubMed ID: 26150390
Summary:
Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. This study investigates the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, it was shown that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. The study identifies an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. These data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle-dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.

Richens, J.H., Barros, T.P., Lucas, E.P., Peel, N., Pinto, D.M., Wainman, A. and Raff, J.W. (2015). The Drosophila Pericentrin-like-protein (PLP) cooperates with Cnn to maintain the integrity of the outer PCM. Biol Open [Epub ahead of print]. PubMed ID: 26157019
Summary:
Centrosomes comprise a pair of centrioles surrounded by a matrix of pericentriolar material (PCM). In vertebrate cells, Pericentrin plays an important part in mitotic PCM assembly, but the Drosophila Pericentrin-like protein (PLP) appears to have a more minor role in mitotic fly cells. This study investigates the function of PLP during the rapid mitotic cycles of the early Drosophila embryo. Unexpectedly, it was found that PLP is specifically enriched in the outer-most regions of the PCM, where it largely co-localizes with the PCM scaffold protein Cnn. In the absence of PLP the outer PCM appears to be structurally weakened, and it rapidly disperses along the centrosomal MTs. As a result, centrosomal MTs are subtly disorganized in embryos lacking PLP, although mitosis is largely unperturbed and these embryos develop and hatch at near-normal rates. Y2H analysis reveals that PLP can potentially form multiple interactions with itself and with the PCM recruiting proteins Asl, Spd-2 and Cnn. A deletion analysis suggests that PLP participates in a complex network of interactions that ultimately help to strengthen the PCM.

Ramdas Nair, A., Singh, P., Salvador Garcia, D., Rodriguez-Crespo, D., Egger, B. and Cabernard, C. (2016). The microcephaly-associated protein Wdr62/CG7337 is required to maintain centrosome asymmetry in Drosophila neuroblasts. Cell Rep [Epub ahead of print]. PubMed ID: 26804909
Summary:
Centrosome asymmetry has been implicated in stem cell fate maintenance in both flies and vertebrates, but the underlying molecular mechanisms are incompletely understood. This study reports that loss of CG7337, the fly ortholog of WDR62, compromises interphase centrosome asymmetry in fly neural stem cells (neuroblasts). Wdr62 maintains an active interphase microtubule-organizing center (MTOC) by stabilizing microtubules (MTs), which are necessary for sustained recruitment of Polo/Plk1 to the pericentriolar matrix (PCM) and downregulation of Pericentrin-like protein (Plp). The loss of an active MTOC in wdr62 mutants compromises centrosome positioning, spindle orientation, and biased centrosome segregation. wdr62 mutant flies also have an approximately 40% reduction in brain size as a result of cell-cycle delays. It is proposed that CG7337/Wdr62, a microtubule-associated protein, is required for the maintenance of interphase microtubules, thereby regulating centrosomal Polo and Plp levels. Independent of this function, Wdr62 is also required for the timely mitotic entry of neural stem cells.

Roque, H., Saurya, S., Pratt, M. B., Johnson, E. and Raff, J. W. (2018). Drosophila PLP assembles pericentriolar clouds that promote centriole stability, cohesion and MT nucleation. PLoS Genet 14(2): e1007198. PubMed ID: 29425198
Summary:
Pericentrin is a conserved centrosomal protein whose dysfunction has been linked to several human diseases. It has been implicated in many aspects of centrosome and cilia function, but its precise role is unclear. This study examined Drosophila Pericentrin-like-protein (PLP) function in vivo in tissues that form both centrosomes and cilia. Plp mutant centrioles exhibit four major defects: (1) They are short and have subtle structural abnormalities; (2) They disengage prematurely, and so overduplicate; (3) They organise fewer cytoplasmic MTs during interphase; (4) When forming cilia, they fail to establish and/or maintain a proper connection to the plasma membrane-although, surprisingly, they can still form an axoneme-like structure that can recruit transition zone (TZ) proteins. PLP helps assemble "pericentriolar clouds" of electron-dense material that emanate from the central cartwheel spokes and spread outward to surround the mother centriole. It is proposed that the partial loss of these structures may largely explain the complex centriole, centrosome and cilium defects observed in Plp mutant cells.
Fang, J. and Lerit, D. A. (2019). Drosophila pericentrin-like protein promotes the formation of primordial germ cells. Genesis: e23347. PubMed ID: 31774613
Summary:
Primordial germ cells (PGCs) are the precursors to the adult germline stem cells that are set aside early during embryogenesis and specified through the inheritance of the germ plasm, which contains the mRNAs and proteins that function as the germline fate determinants. In Drosophila melanogaster, formation of the PGCs requires the microtubule and actin cytoskeletal networks to actively segregate the germ plasm from the soma and physically construct the pole buds (PBs) that protrude from the posterior cortex. Of emerging importance is the central role of centrosomes in the coordination of microtubule dynamics and actin organization to promote PGC development. Previous work has identified a requirement for the centrosome protein Centrosomin (Cnn) in PGC formation. Cnn interacts directly with Pericentrin-like protein (PLP) to form a centrosome scaffold structure required for pericentriolar material recruitment and organization. This study identified a role for PLP at several discrete steps during PGC development. PLP was found to function in segregating the germ plasm from the soma by regulating microtubule organization and centrosome separation. These activities further contribute to promoting PB protrusion and facilitating the distribution of germ plasm in proliferating PGCs.
BIOLOGICAL OVERVIEW

Centrosomes consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Proteins that contain a Pericentrin/AKAP450 centrosomal targeting (PACT) domain have been implicated in recruiting several proteins to the PCM. The only PACT domain protein in Drosophila (the Drosophila pericentrin-like protein [D-PLP]) is associated with both the centrioles and the PCM, and is essential for the efficient centrosomal recruitment of all six PCM components tested. Surprisingly, however, all six PCM components are eventually recruited to centrosomes during mitosis in d-plp mutant cells, and mitosis is not dramatically perturbed. Although viable, d-plp mutant flies are severely uncoordinated, a phenotype usually associated with defects in mechanosensory neuron function. The sensory cilia of these neurons are malformed and the neurons are nonfunctional in d-plp mutants. Moreover, the flagella in mutant sperm are nonmotile. Thus, D-PLP is essential for the formation of functional cilia and flagella in flies (Martinez-Campos, 2004).

Centrosomes are the main microtubule-organizing centers in animal cells, and they consist of a pair of centrioles surrounded by an amorphous pericentriolar material. Proteins that contain a Pericentrin/AKAP450 centrosomal targeting (PACT) domain (Gillingham, 2000) have been implicated in recruiting several proteins to the centrosome. Pericentrin (also called Kendrin; Flory, 2000; Flory, 2003) is a component of the PCM, and anti-pericentrin antibodies disrupt meiotic and mitotic divisions when injected into frog embryos (Doxsey, 1994). The overexpression of Pericentrin in tissue culture cells also leads to mitotic spindle defects (Purohit, 1999; Pihan, 2001). Pericentrin forms a complex with the gamma-tubulin ring complex, and both proteins form a unique structural lattice within the PCM (Dictenberg, 1998). Pericentrin also interacts with cytoplasmic dynein, and this interaction is thought to play a role in the recruitment of Pericentrin and gamma-tubulin to centrosomes (Purohit, 1999; Young, 2000). Thus, it is widely believed that Pericentrin is essential for mitosis. In support of this possibility, SPC110, the only PACT domain protein in Saccharomyces cerevisiae, is an essential protein that tethers the microtubule-nucleating Tub4 (gamma-tubulin in S. cerevisiae) complex to the inner side of the spindle pole body (Knop, 1997; Nguyen, 1998; Martinez-Campos, 2004 and references therein).

The mammalian A-kinase anchoring protein AKAP450 (also called CG-NAP; Takahashi, 1999) contains a PACT domain and recruits PKA and several other proteins to the centrosome (Keryer, 1993, 2003a; Takahashi, 1999). Displacement of the endogenous AKAP450 from centrosomes in tissue culture cells (by overexpression of the AKAP450 PACT domain) leads to defects in cytokinesis, cell cycle progression, and centriole replication (Keryer, 2003a). In these analyses, the displacement of AKAP450 from centrosomes did not disrupt the centrosomal localization of Pericentrin or gamma-tubulin. However, there may be some functional redundancy between Pericentrin and AKAP450/CG-NAP, since Pericentrin can interact with PKA (Diviani, 2000), and AKAP450/CG-NAP can interact with components of the gamma-tubulin ring complex (Takahashi, 2002; Martinez-Campos, 2004 and references therein).

In Drosophila, the predicted gene CG6735 encodes the only recognizable PACT domain protein (Gillingham, 2000), and this protein has been called the Drosophila Pericentrin-like protein (D-PLP). There are two distinct fractions of D-PLP associated with centrosomes -- one that associates with centrioles and another that associates with the PCM. D-PLP is required for the efficient centrosomal recruitment of not just gamma-tubulin, but of all six PCM components tested. Surprisingly, however, all of these PCM components can eventually be recruited to mitotic spindle poles in d-plp mutants, and mutants are viable and exhibit few (if any) mitotic defects. However, mutant flies are severely uncoordinated, a phenotype often associated with defects in mechanosensory neuron function. The mechanosensory cilia in these neurons are abnormal in d-plp mutants, and the cells can no longer respond to external stimuli. Moreover, d-plp mutant sperm are nonmotile, suggesting that D-PLP is essential for the proper function of all cilia and flagella in flies (Martinez-Campos, 2004).

The PACT domain proteins Pericentrin and AKAP450/CG-NAP are among the best-studied centrosomal proteins. These proteins are thought to function by recruiting a small number of specific proteins, such as gamma-tubulin and PKA, to the PCM, and they are widely believed to play important roles in several aspects of cell division (Keryer, 1993; Keryer, 2003a; Doxsey, 1994; Dictenberg, 1998; Takahashi, 1999, 2002; Diviani, 2000; Young, 2000). These analyses on D-PLP, the only PACT domain protein in flies, reveal several important insights into the function of this conserved family of proteins in vivo (Martinez-Campos, 2004).

Previous reports have suggested that Pericentrin and AKAP450 are concentrated in the PCM (Keryer, 1993; Doxsey, 1994; Gillingham, 2000). However, this study found that D-PLP is most strongly associated with the centrioles. Two different anti-D-PLP antibodies stain centrosomes as a very small dot at the center of the PCM, and both antibodies recognize the centrioles in Drosophila oocytes and in interphase larval brain cells -- cells where the centrioles appear to lack any associated PCM. However, FRAP analysis of GFP-PACT in living embryos suggests that there are two distinct fractions of D-PLP at centrosomes: a fraction that stably associates with centrioles (and is only incorporated into centrioles when they replicate) and a fraction in the PCM that is in rapid exchange with a cytoplasmic pool. Although the localization of GFP-PACT may not accurately reflect the localization of the endogenous D-PLP protein, it has been shown that the overexpression of the PACT domain can displace endogenous PACT domain proteins from the centrosome, strongly arguing that this domain binds to the same sites in the centrosome as the endogenous protein (Gillingham, 2000; Keryer, 2003a). Moreover, D-PLP and GFP-PACT colocalize in oocytes, embryos, and larval brain cells in a manner that is distinct from that seen with any other centrosomal markers. Thus, it is thought that GFP-PACT is likely to be a reliable marker of D-PLP localization (Martinez-Campos, 2004).

It is speculated that other PACT domain proteins will also be concentrated in the centrioles as well as in the PCM. Although several previous reports have concluded that these proteins are components of the PCM, some of this data is consistent with a localization in centrioles (Doxsey, 1994; Keryer, 2003a). Pericentrin, for example, is concentrated at the basal body of Xenopus sperm nuclei, whereas other PCM markers, such as gamma-tubulin, are not. In addition, the centriolar fraction of PACT domain proteins may be difficult to detect by indirect immunofluorescence methods in some systems because of antibody penetration problems. In most Drosophila cells, centrioles are unusually small and simple structures, but in spermatocytes they are larger and more elaborate, and more closely resemble the centrioles found in typical vertebrate cells. In Drosophila spermatocytes, neither D-PLP antibody stains the centrioles. This appears to be due to antibody penetration problems, since GFP-PACT is concentrated in these centrioles, but cannot be detected with anti-GFP antibodies (Martinez-Campos, 2004).

D-PLP is essential for the efficient centrosomal recruitment of all six PCM components tested, and no PCM component has been found whose concentration at the centrosome is not perturbed in d-plp mutant cells. This suggests that D-PLP plays an important part in recruiting most (and possibly all) PCM components to the centrosome. However, as mitosis proceeds, the recruitment of PCM components to the centrosome in mutant cells improves, and by the time the cells have entered anaphase, ~80% of cells have a normal centrosomal concentration of PCM markers. A possible explanation is that the d-plp mutations are not nulls, and that a small amount of residual D-PLP function can eventually recruit proteins to centrosomes. However, this is thought unlikely, since 17 alleles of d-plp have been generated, several of which appear to be protein nulls by Western blotting and immunofluorescence criteria. gamma-Tubulin can be detected at centrosomes and mitosis does not appear to be appreciably perturbed in any of these 17 alleles. This suggests that there is a mechanism of recruiting PCM proteins to the centrosomes/spindle poles that is independent of D-PLP (Martinez-Campos, 2004).

How might D-PLP recruit proteins to the centrosome? Observation that D-PLP is associated with both the centrioles and the PCM raises a number of possibilities. D-PLP could function to directly link the centrioles to the PCM: in the absence of D-PLP, no PCM is initially recruited to the centrioles, but a D-PLP–independent mechanism eventually recruits the PCM to the spindle poles as mitosis progresses. Alternatively, the centriolar- and PCM-associated fractions of D-PLP may have separate functions, and D-PLP could interact with PCM components in the cytoplasm and so target them to the centrosome. Finally, D-PLP may play no direct role in recruiting proteins to the PCM, but could simply provide structural integrity to the PCM (Martinez-Campos, 2004).

Perturbing Pericentrin function in Xenopus eggs or in tissue culture cells leads to defects in spindle assembly (Doxsey, 1994; Purohit, 1999; Pihan, 2001), whereas perturbing AKAP450 function leads to defects in cytokinesis, centriole replication, and cell cycle progression (Keryer, 2003a). Recently, the large form of D-PLP (called CP309 in this paper) has been shown to be essential for microtubule nucleation from purified Drosophila centrosomes in vitro (Kawaguchi, 2004). Surprisingly, however, this study found that cell division is not dramatically perturbed in d-plp mutant larval brain cells. Centriole replication appears to occur normally, and even in cells that largely fail to recruit centrosomal proteins to the spindle poles, mitosis occurs relatively normally. This finding is consistent with several previous observations. In cnn and asterless (asl) mutant brain cells, for example, there is a dramatic reduction in the amount of gamma-tubulin recruited to centrosomes during mitosis, but these cells have few mitotic defects. It seems that several cell types can organize bipolar spindles in the absence of centrosomes. Therefore, it is speculated that PACT domain proteins may be dispensable for cell division in vivo in all higher eukaryotic organisms (Martinez-Campos, 2004).

Although D-PLP is not essential for viability, d-plp mutants are invariably uncoordinated, a phenotype often associated with defects in mechanosensory transduction. Type I sensory organs contain sensory neurons with specially modified cilia that transduce proprioceptive and auditory stimuli. Recently, mutations in several genes have been identified that cause defects in cilia formation in these sensory neurons, and these mutations have an uncoordinated phenotype that is very similar to that seen in d-plp mutants. The chordotonal sensory neurons in d-plp mutant flies lack normal cilia and are almost completely nonfunctional, suggesting that cilia defects in the mechanosensory neurons cause the uncoordinated phenotype in d-plp mutants. Moreover, the flagella of sperm cells, which are the only other cells in Drosophila that contain cilia/flagella, are nonmotile, suggesting that D-PLP is required for the proper function of all cilia/flagella in flies (Martinez-Campos, 2004).

What is the essential role of D-PLP in flagella and cilia? It is proposed that D-PLP has a role in maintaining the structural integrity of centrioles/basal bodies in cells that form cilia or flagella. In support of this possibility, the large centrioles formed in d-plp mutant spermatocytes often lose their orthogonal arrangement and partially fragment during development. Such a centriolar 'fragmentation' has not been described before, and it suggests that the structural integrity of the centrioles in these cells is compromised in the absence of D-PLP. In vertebrates, cilia are important in many processes, including sperm motility, sensory neuron function, phototransduction, and the generation of left/right asymmetry during development. It is predicted that knocking out PACT domain protein function in a vertebrate organism would lead to defects in all of these cilia-dependent processes (Martinez-Campos, 2004).

Sperm head-tail linkage requires restriction of pericentriolar material to the proximal centriole end

The centriole, or basal body, is the center of attachment between the sperm head and tail. While the distal end of the centriole templates the cilia, the proximal end associates with the nucleus. Using Drosophila, this study identified a centriole-centric mechanism that ensures proper proximal end docking to the nucleus. This mechanism relies on the restriction of pericentrin-like protein (PLP) and the pericentriolar material (PCM) to the proximal end of the centriole. PLP is restricted proximally by limiting its mRNA and protein to the earliest stages of centriole elongation. Ectopic positioning of PLP to more distal portions of the centriole is sufficient to redistribute PCM and microtubules along the entire centriole length. This results in erroneous, lateral centriole docking to the nucleus, leading to spermatid decapitation as a result of a failure to form a stable head-tail linkage (Galletta, 2020).

An essential element of functional flagellated sperm is proper attachment between the head, which contains the genetic material, and the tail, which generates the force for swimming. A failure in this connection can result in decapitated, decaudated, or malformed sperm, ultimately leading to reduced fertility. The most well documented human study investigated 10 infertile males with acephalic sperm. These patients showed a variety of 'abnormal head-neck configurations,' including breaks between the head and tail and sperm with nuclei laterally attached to the midpiece near the centrioles. The head to tail linkage is centered around the centriole, referred to in this context as a basal body (the term centriole is used for simplicity). The 'distal end' of the centriole templates and anchors the axoneme, the core structural element of the tail, while the 'proximal end' of the centriole forms a connection with the nuclear surface. Thus, the proximal and distal ends of centrioles play distinct and critical roles in sperm assembly. Note that the proximal end and distal end of an individual centriole are being discussed; these are distinct from the 'proximal centriole' and 'distal centriole' terms used in mammalian systems to describe the two centrioles within each sperm. Except when specifically discussing mammalian spermiogenesis, the latter terms will not be used or referenced in this study (Galletta, 2020).

A prerequisite for a tight connection between the head and tail of the sperm is the relocation of the centriole to the nuclear envelope during early spermiogenesis in Drosophila and in mammals. In Drosophila, after the exit from meiosis II, the centriole is repositioned against the reformed nuclear envelope and eventually becomes embedded in the nuclear envelope and is surrounded by an electron-dense material suggested to provide a tight connection. Similarly in mammals, the 'proximal centriole'" moves and attaches to the nucleus where an electron-dense material accumulates and the 'connecting piece' assembles around the centriole pair. In both systems, the centriole templating the flagellar axoneme has its proximal end closest to the nucleus and is positioned perpendicular to the nuclear surface (Galletta, 2020).

In mammalian model systems, there are examples of mutations that cause the head-tail connection to fail, including mutations in centriole proteins Centrin 1 and Centrobin, but little is understood at the molecular level of how these centriole proteins are involved in establishing the head-tail connection. Mutant analysis in Drosophila has identified additional players such as Asunder, Lis-1, Spag4, Yuri gagarin, and dynein and dynactin components, indicating that positioning the microtubule (MT) motor dynein at the nuclear envelope is critical. Finally, mutations in gamma tubulin ring complex proteins, which are required for proper MT formation, result in defective centriole-nuclear attachment in older developing spermatids (STs). It is believed these studies in totality suggest a model whereby dynein on the nuclear surface binds and acts on centriolar MTs to reposition the centriole to the nucleus. However, this process has never been documented in live STs, the role of the centriole itself in this process has not been examined, and the molecular mechanism that ensures correct centriole orientation and docking has not been investigated in detail (Galletta, 2020).

The highly stereotypical proximal end-on docking of centrioles to the nucleus suggests that the centriole proximal end is specialized. Many studies have carefully defined proteins uniquely positioned along the proximal-distal centriole axis. This polarized localization can convey local functions. For example, proteins that regulate centriole length such as Klp10A, Cep97, and Cp110 are positioned at the distal end of the centriole where centriole elongation is thought to exclusively occur. When a new centriole forms, proteins such as Ana2/STIL and Sas6, structural elements required for earliest steps daughter centriole formation, accumulate at the proximal end of the mother centriole. Additionally, pericentriolar material (PCM), which is critical for nucleating and organizing MTs, appears to be restricted to the centriole proximal end in mammalian systems and the meiotic centrioles of Drosophila spermatocytes (SC). This suggests that MTs are predominantly produced and anchored at the proximal end of centrioles. However, unlike distal-end protein components, the importance of restricting PCM to the proximal end and the mechanism by which this restriction is achieved are unknown. This study sought to identify the mechanism by which PCM is proximally restricted, and it was hoped, in turn, to gain insight into proximal end nuclear docking during spermatogenesis (Galletta, 2020).

Functional sperm require a stable linkage between sperm head and tail, which is mediated by the centriole. To date, little is known about the molecular mechanisms underlying defective head-tail attachment, except for a few reports identifying mutations in genes such as Spata6, Sun5, BRDT, PMFBP1, and TSGA10. Directly relevant to this study on the role of the centriole in head-tail attachment, is TSGA10 (Cep135 paralog) and two other centriole proteins -- Centrin1 and Centrobin. Thus, the limited patient sequence analysis and mammalian model system mutants point to a critical role for the centriole in head-tail attachment (Galletta, 2020).

Live imaging of centrioles in Drosophila STs revealed that the docking of the centriole to the nucleus is a two-step process. The first is 'Nuclear Search,' where the centriole searches for its docking partner, the nucleus, immediately following meiotic exit. The second is 'Nuclear Attachment,' where a stable connection of the centriole to the nuclear surface is formed. Work in Drosophila has also identified a number of mutations that ultimately result in decapitation, several of which affect the ability of the MT motor dynein to localize to the NE, or to generate pulling forces on MTs. Interestingly, studies from mice have also implicated the dynein adapter Hook1 and the sun protein SPAG4 in ensuring proper head-tail linkage. These proteins have been shown across species to position the centrosomes adjacent to the nucleus in interphase of normally cycling cells. Thus, a simple model emerges where in developing STs a conserved dynein-based system on the surface of the nucleus interacts with MTs emanating specifically from the proximal end of the centriole to draw the proximal centriole end specifically to the nuclear surface. Imaging of MTs in Drosophila suggests that this model is quite plausible, showing MTs specifically emerge from the proximal end of the centriole, which was then shown is key to proper centriole-nuclear docking. One exciting future direction is to reexamine the known Drosophila decapitation mutants to separate players that affect Nuclear Search from those that affect only the later step of Nuclear Attachment (Galletta, 2020).

While this study focuses on the formation of functional sperm, it also provides insight into centrosome architecture and how this architecture relates to function, in this case nuclear docking. In recent years, enormous progress has been made in identifying centrosome proteins and mapping their localization with nanometer precision. The challenge now is to link protein position with function; progress on this front has been most notably made at the centriole. For example Ana2/STIL, Sas6, and Cep135 form a cartwheel structure inside a new centriole at its proximal end, serving as a template for centriole symmetry. Another protein complex, Cep97, CP110, and Klp10A, localizes to the distal end and to control centriole length. Thus, subcentriolar localization and function are intimately linked. Despite PCM being documented at the proximal end of the centriole, a link between the position and function of the PCM at the proximal end has not been established. One possible role for the PCM at the proximal end is to dictate the position of daughter centriole formation. Previous work has shown that new daughter centriole nucleation requires PCM, and overexpression of PCM results in additional daughter centriole formation. However, the position of the ectopic centrioles along the proximal-distal axis was not examined, and the importance of the proximal position could not be inferred. This study provides a clear demonstration of a proximal-specific function for PCM (Galletta, 2020).

Through a series of wild-type, mutant, and misexpression experiments, this study showed that the bridge protein PLP is critical in proper centriole docking in STs. By examining plp mutant testes, it was found that PLP is a major driver of PCM recruitment or retainment at centrioles in round spermatids (RSTs). In the absence of PLP, PCM is disorganized and the centriole is improperly positioned away from the nucleus. This study then investigated how PLP itself is restricted to the proximal end. Using endogenously tagged PLP protein, single-cell RNA-seq and whole mount in situ, this study showed that proximal restriction of PLP is achieved through a reduction in PLP mRNA and protein concentration prior to centriole elongation. It will be important in future studies to determine precisely how PLP concentration is reduced; there is likely a delicate balance between decreased PLP translation, increased PLP degradation, and simple dilution of PLP as spermatocyte size increases (Galletta, 2020).

While numerous studies have shown that PLP is necessary for PCM organization around centrioles and that PLP can interact with PCM components, the precise mechanism of how PLP acts to recruit or anchor γ-tub is not known. In addition to interacting with PCM components, both PLP, and its mammalian ortholog pericentrin, have been found in complexes with γ-tub itself. It remains to be determined precisely how the multiple possible pathways through which PLP can affect the PCM function at the centriole in different contexts. Whether direct or indirect, this study shows that mislocalizing PLP on the centriole is sufficient to dictate PCM position on the centriole. When PCM is present at a more distal position along the centriole in STs, MTs emanate from the entire centriole, which can result in lateral capture to the nucleus. This lateral capture appears unstable and firm Nuclear Attachment does not occur. It is proposed that these failed, or defective, centriole-nuclear attachments do not survive the forces applied as a result of axoneme and mitochondrial derivative elongation, nuclear clustering, and/or ST individualization, ultimately resulting in sperm decapitation. This is consistent with several studies linking a failure in individualization to a failure in centriole-nuclear attachment. Additional studies will be required to determine how the tight attachment between the nucleus and the centriole forms, precisely when the Nuclear Attachment fails in STs with laterally docked centrioles and how this connection relates to the machinery that drives the massive cellular reorganization required to build sperm (Galletta, 2020).

The transcriptional mechanism to restrict protein localization this study identified is one way to achieve sub-centriolar protein compartments. Other mechanisms include specific docking-site recognition, such as the LID (longitudinal tubulin-tubulin interaction domain) domain of Sas4 recognizing the plus end of MTs at the centriole distal end and protein symmetry-breaking and coalescence as seen with Plk4's ability to concentrate into a single spot on the surface of the centriole. While it is unknown if other proteins use a transcriptional mechanism like PLP for centriole position control, single-cell RNA-seq data could help identified such proteins (Galletta, 2020).

One exciting finding from this work is that simply altering the timing of PLP expression can have major deleterious effects. This can be analogous to many human diseases that are frequently reported to correlate with higher levels of protein expression; understanding the underlying cell biology and physiology of the protein overexpression becomes quite critical. For example, overexpression of the master regulator of centriole duplication Plk4 is sufficient to promote tumorigenesis and renal cysts. Another example is seen in the case of having an extra copy of pericentrin (the ortholog of PLP), which in humans is present on chromosome 21. An increase in pericentrin protein levels by 50% can cause defects in ciliogenesis and cilia function. Therefore, understanding the role of the centriole in human disorders will not only require understanding the consequences of loss of protein function but also the consequence of protein misexpression and misregulation (Galletta, 2020)


GENE STRUCTURE

cDNA clone length - 9125

Exons - 5

Bases in 3' UTR - 431

PROTEIN STRUCTURE

Amino Acids - 2726

Structural Domains

It was reasoned that CaM-binding proteins such as Kendrin and CG-NAP, that are important for centrosome function, should exist in Drosophila. Database searches using the full-length Kendrin or CG-NAP did not reveal any Drosophila proteins sharing overall sequence homology with Kendrin or CG-NAP. However, several predicted Drosophila proteins were found to contain the highly conserved CaM-binding motif that is found in Spc110, Kendrin, and CG-NAP. Interestingly, only one of these proteins, encoded by CG6735, is predicted to contain largely coiled-coil sequences 5' to the C-terminal CaM-binding domain such as Spc110, Kendrin, and CG-NAP. Because CG6735 encodes a potential homolog of Kendrin and CG-NAP, the gene product of CG6735 was analyzed further (Kawaguchi, 2004).

Sequence analyses reveal that CG6735 encodes a complete open reading frame of 1109 amino acids. This predicted protein is significantly shorter than Kendrin (3246 amino acids) and CG-NAP (3899 amino acids). Therefore, it was asked whether there is a longer splicing variant(s) of CG6735 in Drosophila by using RACE analyses starting from the 5' end of CG6735. This led to the identification of a complete open reading frame that encodes a predicted protein of 2726 amino acids and 309 kDa. This protein will be referred to as CP309 (Kawaguchi, 2004).

CP309 is similar to Kendrin and CG-NAP in the following three aspects: (1) all three proteins contain the conserved pericentrin AKAP450 centrosome targeting (PACT) domain of 200 amino acids at their C termini; (2) they have similar coiled-coil organizations N-terminal to the PACT domain, and (3) they share a conserved region of 40 amino acids located in the N-terminal half of these proteins. Based on these analyses, it is suggested that CP309 is the Drosophila equivalent of Kendrin or CG-NAP. Although CP309 can bind to CaM in the absence of Ca2+, Ca2+ significantly enhances the binding (Kawaguchi, 2004).

The predicted protein encoded by the CG6735 gene contains a COOH-terminal PACT domain. The COOH-terminal 226 amino acids of this protein fused to GFP localizes to centrosomes when ectopically expressed in human tissue culture cells (Gillingham, 2000). A full-length cDNA has been isolated for CG6735 (D-PLP-S), and recently a longer cDNA has been isolated that incorporates two other predicted genes in this region, D-PLP-L; Kawaguchi, 2004). Thus, the d-plp gene encodes at least one large (D-PLP-L) and one small (D-PLP-S) form of D-PLP (Martinez-Campos, 2004).


Pericentrin-like protein: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 25 July 2006

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