Gene name - Pericentrin-like protein
Synonyms - D-PLP
Cytological map position - 71B2--4
Function - scaffolding protein
Symbol - Plp
FlyBase ID: FBgn0086690
Genetic map position - 3-
Classification - PACT domain protein
Cellular location - cytoplasmic/centriolar
|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
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
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
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
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.
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-PLPindependent 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).
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).
date revised: 25 July 2006
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