<i>Drosophila</i>pericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies

Galletta, Brian J.; Guillen, Rodrigo X.; Fagerstrom, Carey J.; Brownlee, Christopher; Lerit, Dorothy A.; Megraw, Timothy L.; Rogers, Gerald S.; Rusan, Nasser M.

doi:10.1091/mbc.e13-10-0617

Cited by 44 publications

(87 citation statements)

References 57 publications

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“…Our attempts to detect CN enrichment at centrosomes have been unsuccessful (data not shown) and may indicate that CN-centrosome interactions are limited, transient, and/or temporally regulated. Investigations of Ca 2+ signaling at centrosomes, which contain multiple Ca 2+ -and/or calmodulin-binding proteins (Dantas et al, 2013;Galletta et al, 2014;Spektor et al, 2007;Tsang et al, 2006) and cilia, which constitute a distinct Ca 2+ compartment (Delling et al, 2013;Delling et al, 2016), are ongoing. Our findings point to CN as a likely downstream effector of Ca 2+ signals at these organelles.…”

Section: Discussionmentioning

confidence: 99%

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Wigington

Roy

Damle

et al. 2019

Preprint

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Short linear motifs (SLiMs) form dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN), the Ca 2+ -regulated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in situ SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including Notch1, which we establish as a CN substrate. Unexpectedly, CN shows SLiMdependent proximity to centrosomal and nuclear pore complex (NPC) proteins -structures where Ca 2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca 2+ and CN signaling and the demonstrated synergy between experimental and computational methods establishes a blueprint for examining SLiM-based networks.

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Section: Discussionmentioning

confidence: 99%

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Wigington

Roy

Damle

et al. 2019

Preprint

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“…One such modification was to make the Y2H vectors pGBKT7 and pGADT7 compatible with the Gateway cloning system (Rossignol et al ., 2007); Life Technologies, Grand Island, NY). Our lab has further modified the Gateway compatible pGBKT7 vector by replacing the kanamycin resistance cassette with one providing resistance against ampicillin so that it could be used with Gateway Entry clones (Galletta et al ., 2014). Sequences encoding the fragments should be generated by PCR and then cloned into Entry vectors.…”

Section: Performing An Array Based Screen For Centrosome Protein Imentioning

confidence: 99%

A yeast two-hybrid approach for probing protein–protein interactions at the centrosome

Galletta¹,

Rusan²

2015

Centrosome &Amp; Centriole

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As a large, non-membrane bound organelle, the centrosome must rely heavily on protein-protein interactions to assemble itself in the cytoplasm and perform its functions as a microtubule-organizing center. Therefore, to understand how this organelle is built and functions, one must understand the protein-protein interactions made by each centrosome protein. Unfortunately, the highly interconnected nature of the centrosome, combined with its predicted unstructured, coil-rich proteins, has made the use of many standard approaches to studying protein-protein interactions very challenging. The yeast-two hybrid (Y2H) system is well suited for studying the centrosome and is an important complement to other biochemical approaches. In this chapter we describe how to carry out a directed Y2H screen to identify the direct interactions between a given centrosome protein and a library of others. Specifically, we detail using a bioinformatics based approach (structure prediction programs) to subdivide proteins and screen for interactions using an array-based Y2H approach. We also describe how to use the interaction information garnered from this screen to generate mutations to disrupt specific interactions using mutagenic-PCR and a “reverse” Y2H screen. Finally, we discuss how information from such a screen can be integrated into existing models of centrosome assembly and how it can initiate and guide extensive in vitro and in vivo experimentation to test these models.

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“…We studied its consequences in the localization of basal body components and basal body structure and, subsequently, in male fertility ( Figure 7A). Unlike the plp mutant flies (Martinez-Campos et al, 2004;Galletta et al, 2014), the knockdown of PLP by RNAi did not affect centriole number in sperm cells ( Figure 7B, C). Remarkably, the RNAi affected SAS-6 recruitment to the BBs (Figure 7D, E).…”

Section: Drosophila Pericentrin Is Required For Sas-6 Recruitment Andmentioning

confidence: 85%

“…The PACT domain contains two highly conserved calmodulin (CaM)-binding domains (CBD1 and CBD2) (Galletta et al, 2014, Figure 8A). The interaction of pericentrin with calmodulin at CaM-binding domain is conserved and is important for its function both in animals and yeasts.…”

Section: Drosophila Pericentrin Is Required For Sas-6 Recruitment Andmentioning

confidence: 99%

An ancestral role of pericentrin in centriole formation through SAS-6 recruitment

Ito

Zitouni

Jana

et al. 2018

Preprint

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Short summaryThe pericentriolar matrix (PCM) is not only important for microtubule-nucleation but also can regulate centriole biogenesis. Ito et al. reveal an ancestral interaction between the centriole protein SAS-6 and the PCM component pericentrin, which regulates centriole biogenesis and elongation. AbstractThe centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.

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Drosophilapericentrin requires interaction with calmodulin for its function at centrosomes and neuronal basal bodies but not at sperm basal bodies

Cited by 44 publications

References 57 publications

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling

A yeast two-hybrid approach for probing protein–protein interactions at the centrosome

An ancestral role of pericentrin in centriole formation through SAS-6 recruitment

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