Centrosome Duplication and Maturation

National Heart, Lung, And Blood Institute

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Abstract

As a non-membrane bound organelle, the assembly of centrosomes must be driven by PPIs. These interactions are likely modified by highly regulated changes in protein binding affinity in a cell-type and cell-cycle dependent manner, which can in turn modulate centrosome behavior and function. We have focused on identifying PPIs among a core set of conserved centrosome proteins, classified as proteins of the centriole, the PCM and regulatory kinases. Previous high-throughput and small-scale interactions studies have suggested that there might be a limited number of PPIs among centrosome proteins and have raised the possibility that few interactions are used to construct a simple, ultimately static structure. Our data does not support this simplistic view of the centrosome. We have uncovered a large number of direct PPIs, dramatically expanding our understanding of the centrosome interaction landscape. This highly interconnected landscape suggests a much more complex centrosome assembly and regulatory process, which we suggest can be leveraged to perform a variety of specialized tasks dictated by a broad spectrum of cellular requirements. In previous years we have focused our attention on Pericentrin-Like-Protein (PLP), Asterless (Asl), Cep135, and the critical centriole duplication kinase Plk4. In the past year, we have focused on the role of Ana2 and Sas4 in regulating Plk4. In a collaborative effort with the lab of Greg Rogers at the University of Arizona, we showed that one of the earliest stages of centriole duplication begins by the binding of Ana2 to the Sas4 G-box, when then enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation. Thus, this study discovered a conserved and earliest step in centriole assembly. Key to uncovering the interaction of Ana2 and Sas4, was a novel screen developed in our lab to identify proteins that form stable binary complexes. 17 centrosome genes were selected for analysis, including Sas4, Plk4, and Ana2. FL genes and fragments were randomly inserted into a bacterial dual-tag expression plasmid where one component is tagged with either His6 and the other with GFP. In brief, bacterial lysates were subjected to a two-step affinity-purification scheme to isolate protein complexes. From 992 isolated bacterial colonies, we identified 90 bacterial lysates containing both His6 and GFP proteins. False-positive dual-tagged single proteins were eliminated by PCR analysis, as well as duplicate dual protein expression vectors, leaving a possible 48 positive interactors. Binding was further tested by scaling up the cultures and using a secondary screen consisting of tandem His6 pulldown followed by GFP IP, resulting in 21 positive interactions. These interactions were subjected to a final GFP IP step to test for reciprocal binding, yielding six high-confidence interactors. These included (1) the central Plk4 Polo box (PB) cassette PB1-PB2 with itself, (2) PB1-PB2 and the N terminus of Asl, and (3) NT and C-terminal fragments of Ana2; all three are well-described interactions that validated our screen. Remarkably, we also identified the Sas4 G-box as another PB1-PB2 binding partner as well as an interaction between the G-box and a C-terminal fragment of Ana2 lacking the previously described G-boxbinding motif. (An interaction between the Ana2 N terminus and Cnn was also detected but is not examined further in this study.) We note that G-box/PB1-PB2 binding was not preserved during the tandem GFP-IP step of the screen, indicating that this interaction is weaker compared with the other interactors. This novel screen or randomly assembled complexes continues to guide additional project on centriole assembly in our lab.