Shirin Bahmanyar

Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by ϵ-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.

Regulated assembly of a supramolecular centrosome scaffold in vitro

A little bit of this and a little bit of that Centrosomes are the major microtubule-organizing centers in animal cells. Key to this function is the somewhat mysterious pericentriolar material (PCM). Woodruff et al. describe the in vitro reconstitution of PCM assembly. In cells, PCM is recruited by centrioles to form centrosomes that nucleate and anchor microtubules. SPD-5, the main component of the PCM matrix in Caenorhabiditis elegans, polymerized in vitro to form micrometer-sized porous networks. SPD-5 polymerization was directly controlled by the polo family kinase Plk1 and Cep192/SPD-2, two conserved regulators that control PCM assembly across metazoans. Science, this issue p. 808 Centrosome assembly in Caenorhabditis elegans involves self-assembly of an interconnected, micrometer-scale network of proteins. The centrosome organizes microtubule arrays within animal cells and comprises two centrioles surrounded by an amorphous protein mass called the pericentriolar material (PCM). Despite the importance of centrosomes as microtubule-organizing centers, the mechanism and regulation of PCM assembly are not well understood. In Caenorhabditis elegans, PCM assembly requires the coiled-coil protein SPD-5. We found that recombinant SPD-5 could polymerize to form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Only the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other PCM proteins. Thus, PCM size and binding capacity emerge from the regulated polymerization of one coiled-coil protein to form a porous network.

Nuclear Envelope Phosphatase 1-Regulatory Subunit 1 (Formerly TMEM188) Is the Metazoan Spo7p Ortholog and Functions in the Lipin Activation Pathway

Background: Lipins are phosphatidic acid phosphatases. In yeast, lipin is activated by the Nem1p-Spo7p complex. There is controversy as to whether a mammalian Spo7p ortholog exists. Results: The metazoan Spo7p ortholog is now identified and shown to interact with lipins in yeast, nematodes, and mammalian cells. Conclusion: NEP1-R1 is the metazoan Spo7p ortholog. Significance: The lipin activation system is conserved in evolution. Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly “dullard”), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.

Spatial control of phospholipid flux restricts endoplasmic reticulum sheet formation to allow nuclear envelope breakdown

The nuclear envelope is a subdomain of the endoplasmic reticulum (ER). Here we characterize CNEP-1 (CTD [C-terminal domain] nuclear envelope phosphatase-1), a nuclear envelope-enriched activator of the ER-associated phosphatidic acid phosphatase lipin that promotes synthesis of major membrane phospholipids over phosphatidylinositol (PI). CNEP-1 inhibition led to ectopic ER sheets in the vicinity of the nucleus that encased the nuclear envelope and interfered with nuclear envelope breakdown (NEBD) during cell division. Reducing PI synthesis suppressed these phenotypes, indicating that CNEP-1 spatially regulates phospholipid flux, biasing it away from PI production in the vicinity of the nuclear envelope to prevent excess ER sheet formation and NEBD defects.

Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase

ABSTRACT Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G1 accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.

Role of APC and its binding partners in regulating microtubules in mitosis.

Adenomatous polyposis coli (APC) is a multifunctional protein commonly mutated in colon cancer. APC contains binding sites for multiple proteins with diverse roles in signaling and the structural and functional organization of cells. Recent evidence suggests roles for APC and some of its binding partners in regulating microtubules in mitosis. APC localizes to three key locations in mitosis: kinetochores, the cortex and centrosomes. Here, we discuss possible mechanisms for APC function at these sites and suggest new pathways by which APC mutations promote tumorigenesis.

Formation of extra centrosomal structures is dependent on β-catenin

β-Catenin has important roles in cell–cell adhesion and in the regulation of gene transcription. Mutations that stabilize β-catenin are common in cancer, but it remains unclear how these mutations contribute to cancer progression. β-Catenin is also a centrosomal component involved in centrosome separation. Centrosomes nucleate interphase microtubules and the bipolar mitotic spindle in normal cells, but their organization and function in human cancers are abnormal. Here, we show that expression of stabilized mutant β-catenin, which mimics mutations found in cancer, results in extra non-microtubule nucleating structures that contain a subset of centrosome proteins including γ-tubulin and centrin, but not polo-like kinase 4 (Plk4), SAS-6 or pericentrin. A transcriptionally inactive form of β-catenin also gives rise to abnormal structures of centrosome proteins. HCT116 human colon cancer cell lines, from which the mutant β-catenin allele has been deleted, have reduced numbers of cells with abnormal centrosome structures and S-phase-arrested, amplified centrosomes. RNAi-mediated depletion of β-catenin from centrosomes inhibits S-phase-arrested amplification of centrosomes. These results indicate that β-catenin is required for centrosome amplification, and mutations in β-catenin might contribute to the formation of abnormal centrosomes observed in cancers.

Affinity Purification of Protein Complexes in C. elegans

C. elegans is a powerful metazoan model system to address fundamental questions in cell and developmental biology. Research in C. elegans has traditionally focused on genetic, physiological, and cell biological approaches. However, C. elegans is also a facile system for biochemistry: worms are easy to grow in large quantities, the functionality of tagged fusion proteins can be assessed using mutants or RNAi, and the relevance of putative interaction partners can be rapidly tested in vivo. Combining biochemistry with function-based genetic and RNA interference screens can rapidly accelerate the delineation of protein networks and pathways in diverse contexts. In this chapter, we focus on two strategies to identify protein-protein interactions: single-step immunoprecipitation and tandem affinity purification. We describe methods for growth of worms in large-scale liquid culture, preparation of worm and embryo extracts, immunoprecipitation, and tandem affinity purification. In addition, we describe methods to test specificity of antibodies, strategies for optimizing starting material, and approaches to distinguish specific from non-specific interactions.

Spatial regulation of phospholipid synthesis within the nuclear envelope domain of the endoplasmic reticulum

The endoplasmic reticulum (ER) is an extensive membrane system that serves as a platform for de novo phospholipid synthesis. The ER is partitioned into distinct functional and structural domains, the most notable of which is the nuclear envelope. Here we discuss the role of nuclear envelope localized CNEP-1Nem1 in spatial regulation of de novo phospholipid synthesis within the ER. CNEP-1Nem1 is an activator of lipinPah1, which is the key phosphatidic acid phosphatase that regulates the metabolic branch-point between the production of phosphatidylinositol (PtdIns) and major membrane phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). CNEP-1 activates lipin at the nuclear envelope to bias phospholipid flux toward PC and PE production and to limit PtdIns incorporation. Increased PtdIns causes the formation of ectopic ER sheets in the vicinity of the nucleus that wrap around the nuclear envelope and cause downstream defects in NE disassembly. We propose that spatial regulation of phospholipid flux promotes partitioning of the ER into distinct subdomains by generating a gradient of PtdIns incorporation.

A novel small molecule that disrupts a key event during the oocyte-to-embryo transition in C. elegans

The complex cellular events that occur in response to fertilization are essential for mediating the oocyte-to-embryo transition. Here, we describe a comprehensive small-molecule screen focused on identifying compounds that affect early embryonic events in Caenorhabditis elegans. We identify a single novel compound that disrupts early embryogenesis with remarkable stage and species specificity. The compound, named C22, primarily impairs eggshell integrity, leading to osmotic sensitivity and embryonic lethality. The C22-induced phenotype is dependent upon the upregulation of the LET-607/CREBH transcription factor and its candidate target genes, which primarily encode factors involved in diverse aspects of protein trafficking. Together, our data suggest that in the presence of C22, one or more key components of the eggshell are inappropriately processed, leading to permeable, inviable embryos. The remarkable specificity and reversibility of this compound will facilitate further investigation into the role and regulation of protein trafficking in the early embryo, as well as serve as a tool for manipulating the life cycle for other studies such as those involving aging. Summary: The small molecule C22 induces expression of the LET-607 transcription factor, leading to mis-regulation of protein trafficking and thus impairing eggshell formation and the oocyte-to-embryo transition.