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EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.

Multiple expansion microscopy approaches have been successfully used in the analysis of centrioles, centrosomes, and cilia, helping to reveal the localization of numerous centrosomal and ciliary proteins at nanoscale resolution. In this chapter, we describe the use of two stable STED dyes in combination with expansion microscopy, which allows the robust detection by conventional and STED microscopy of proteins immunolabeled prior to sample expansion. We demonstrate the stability of these dyes during the crosslinking, polymerization, and denaturation steps of an expansion protocol thereby allowing their use in an immunolabel-first-expand-later approach. Our protocol overcomes the frequent technical limitation of poor, unreproducible binding of primary antibodies to proteins after denaturation. We demonstrate the applicability of this approach by analyzing both a centriole appendage protein Cep164 and a ciliary protein ARL13B., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets., (© 2023 Sullenberger et al.)

Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by coexpression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two noncanonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER-resident proteins, including at sites that could escape analyses based on the established consensus sequence., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms underlying male infertility are poorly understood. Most mammalian spermatozoa have two centrioles: the typical barrel-shaped proximal centriole (PC) and the atypical fan-like distal centriole (DC) connected to the axoneme (Ax). These structures are essential for fertility. However, the relationship between centriole quality and subfertility (reduced fertility) is not well established. Here, we tested the hypothesis that assessing sperm centriole quality can identify cattle subfertility. By comparing sperm from 25 fertile and 6 subfertile bulls, all with normal semen analyses, we found that unexplained subfertility and lower sire conception rates (pregnancy rate from artificial insemination in cattle) correlate with abnormal centriolar biomarker distribution. Fluorescence-based Ratiometric Analysis of Sperm Centrioles (FRAC) found only four fertile bulls (4/25, 16%) had positive FRAC tests (having one or more mean FRAC ratios outside of the distribution range in a group's high-quality sperm population), whereas all of the subfertile bulls (6/6, 100%) had positive FRAC tests (P = 0.00008). The most sensitive biomarker was acetylated tubulin, which had a novel labeling pattern between the DC and Ax. These data suggest that FRAC and acetylated tubulin labeling can identify bull subfertility that remains undetected by current methods and may provide insight into a novel mechanism of subfertility., (© 2023. The Author(s).)

We report systematic analysis of endogenous EWSR1's cellular organization. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Interestingly, EWSR1 and FUS, another FET protein, exhibit distinct spatial organizations. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease., Competing Interests: The authors declare no competing financial interests.

Regulated protein degradation in eukaryotes is performed by the 26S proteasome, which contains a 19-subunit regulatory particle (RP) that binds, processes, and translocates substrates to a 28-subunit hollow core particle (CP) where proteolysis occurs. In addition to its intrinsic subunits, myriad proteins interact with the proteasome transiently, including factors that assist and/or regulate its degradative activities. Efforts to identify proteasome-interacting components and/or to solve its structure have relied on over-expression of a tagged plasmid, establishing stable cell lines, or laborious purification protocols to isolate native proteasomes from cells. Here, we describe an engineered human cell line, derived from colon cancer HCT116 cells, with a biotin handle on the RP subunit hRpn1/PSMD2 (proteasome 26S subunit, non-ATPase 2) for purification of 26S proteasomes. A 75-residue sequence from Propionibacterium shermanii that is biotinylated in mammalian cells was added following a tobacco etch virus protease cut site at the C terminus of hRpn1. We tested and found that 26S proteasomes can be isolated from this modified HCT116 cell line by using a simple purification protocol. More specifically, biotinylated proteasomes were purified from the cell lysates by using neutravidin agarose resin and released from the resin following incubation with tobacco etch virus protease. The purified proteasomes had equivalent activity in degrading a model ubiquitinated substrate, namely ubiquitinated p53, compared to commercially available bovine proteasomes that were purified by fractionation. In conclusion, advantages of this approach to obtain 26S proteasomes over others is the simple purification protocol and that all cellular proteins, including the tagged hRpn1 subunit, remain at endogenous stoichiometry., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Mitochondria are essential organelles whose proteome is well protected by regulated protein degradation and quality control. While the ubiquitin-proteasome system can monitor mitochondrial proteins that reside at the mitochondrial outer membrane or are not successfully imported, resident proteases generally act on proteins within mitochondria. Herein, we assess the degradative pathways for mutant forms of three mitochondrial matrix proteins (mas1-1HA, mas2-11HA, and tim44-8HA) in Saccharomyces cerevisiae. The degradation of these proteins is strongly impaired by loss of either the matrix AAA-ATPase (m-AAA) (Afg3p/Yta12p) or Lon (Pim1p) protease. We determine that these mutant proteins are all bona fide Pim1p substrates whose degradation is also blocked in respiratory-deficient "petite" yeast cells, such as in cells lacking m-AAA protease subunits. In contrast, matrix proteins that are substrates of the m-AAA protease are not affected by loss of respiration. The failure to efficiently remove Pim1p substrates in petite cells has no evident relationship to Pim1p maturation, localization, or assembly. However, Pim1p's autoproteolysis is intact, and its overexpression restores substrate degradation, indicating that Pim1p retains some functionality in petite cells. Interestingly, chemical perturbation of mitochondria with oligomycin similarly prevents degradation of Pim1p substrates. Our results demonstrate that Pim1p activity is highly sensitive to mitochondrial perturbations such as loss of respiration or drug treatment in a manner that we do not observe with other proteases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Centrioles are structures that assemble centrosomes. CPAP is critical for centrosome assembly, and its mutations are found in patients with diseases such as primary microcephaly. CPAP's centrosomal localization, its dynamics, and the consequences of its insufficiency in human cells are poorly understood. Here we use human cells genetically engineered for fast degradation of CPAP, in combination with superresolution microscopy, to address these uncertainties. We show that three independent centrosomal CPAP populations are dynamically regulated during the cell cycle. We confirm that CPAP is critical for assembly of human centrioles, but not for recruitment of pericentriolar material on already assembled centrioles. Further, we reveal that CPAP insufficiency leads to centrioles with incomplete microtubule triplets that can convert to centrosomes, duplicate, and form mitotic spindle poles, but fragment owing to loss of cohesion between microtubule blades. These findings further our basic understanding of the role of CPAP in centrosome biogenesis and help understand how CPAP aberrations can lead to human diseases., (© 2022 Vásquez Limeta et al.)

Centrosomes were first described by Edouard Van Beneden and named and linked to chromosome segregation by Theodor Boveri around 1870. In the 1960-1980s, electron microscopy studies have revealed the remarkable ultrastructure of a centriole -- a nine-fold symmetrical microtubular assembly that resides within a centrosome and organizes it. Less than two decades ago, proteomics and genomic screens conducted in multiple species identified hundreds of centriole and centrosome core proteins and revealed the evolutionarily conserved nature of the centriole assembly pathway. And now, super resolution microscopy approaches and improvements in cryo-tomography are bringing an unparalleled nanoscale-detailed picture of the centriole and centrosome architecture. In this chapter, we summarize the current knowledge about the architecture of human centrioles. We discuss the structured organization of centrosome components in interphase, focusing on localization/function relationship. We discuss the process of centrosome maturation and mitotic spindle pole assembly in centriolar and acentriolar cells, emphasizing recent literature., (Published by Elsevier Ltd.)

Centrosomes are composed of a centriolar core surrounded by pericentriolar material that nucleates microtubules. The ubiquitin ligase TRIM37 localizes to centrosomes, but its centrosomal roles are not yet defined. We show that TRIM37 does not control centriole duplication, structure, or the ability of centrioles to form cilia but instead prevents assembly of an ectopic centrobin-scaffolded structured condensate that forms by budding off of centrosomes. In ∼25% of TRIM37-deficient cells, the condensate organizes an ectopic spindle pole, recruiting other centrosomal proteins and acquiring microtubule nucleation capacity during mitotic entry. Ectopic spindle pole-associated transient multipolarity and multipolar segregation in TRIM37-deficient cells are suppressed by removing centrobin, which interacts with and is ubiquitinated by TRIM37. Thus, TRIM37 ensures accurate chromosome segregation by preventing the formation of centrobin-scaffolded condensates that organize ectopic spindle poles. Mutations in TRIM37 cause the disorder mulibrey nanism, and patient-derived cells harbor centrobin condensate-organized ectopic poles, leading us to propose that chromosome missegregation is a pathological mechanism in this disorder., (© 2021 Meitinger et al.)

Centriole copy number is tightly maintained by the once-per-cycle duplication of these organelles. Centrioles constitute the core of centrosomes, which organize the microtubule cytoskeleton and form the poles of the mitotic spindle. Centrosome amplification is frequently observed in tumors, where it promotes aneuploidy and contributes to invasive phenotypes. In non-transformed cells, centrosome amplification triggers PIDDosome activation as a protective response to inhibit cell proliferation, but how extra centrosomes activate the PIDDosome remains unclear. Using a genome-wide screen, we identify centriole distal appendages as critical for PIDDosome activation in cells with extra centrosomes. The distal appendage protein ANKRD26 is found to interact with and recruit the PIDDosome component PIDD1 to centriole distal appendages, and this interaction is required for PIDDosome activation following centrosome amplification. Furthermore, a recurrent ANKRD26 mutation found in human tumors disrupts PIDD1 localization and PIDDosome activation in cells with extra centrosomes. Our data support a model in which ANKRD26 initiates a centriole-derived signal to limit cell proliferation in response to centrosome amplification., (© 2020 The Authors.)

Expansion microscopy is an imaging method based on isotropic physical expansion of biological samples, which improves optical resolution and allows imaging of subresolutional cellular components by conventional microscopes. Centrioles are small microtubule-based cylindrical structures that build centrosomes and cilia, two organelles essential for vertebrates. Due to a centriole's small size, electron microscopy has traditionally been used to study centriole length and ultrastructural features. Recently, expansion microscopy has been successfully used as an affordable and accessible alternative to electron microscopy in the analysis of centriole and cilia length and structural features. Here, we describe an expansion microscopy approach for the analysis of centrioles and cilia in large populations of mammalian adherent and nonadherent cells and multiciliated cultures.

Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.

Centrioles are precisely built microtubule-based structures that assemble centrosomes and cilia. Aberrations in centriole structure are common in tumors, yet how these aberrations arise is unknown. Analysis of centriole structure is difficult because it requires demanding electron microscopy. Here we employ expansion microscopy to study the origins of centriole structural aberrations in large populations of human cells. We discover that centrioles do not have an elongation monitoring mechanism, which renders them prone to over-elongation, especially during prolonged mitosis induced by various factors, importantly including supernumerary centrioles. We identify that mitotic centriole over-elongation is dependent on mitotic Polo-like kinase 1, which we uncover as a novel regulator of centriole elongation in human cycling cells. While insufficient Plk1 levels lead to the formation of shorter centrioles lacking a full set of microtubule triplets, its overactivity results in over-elongated and structurally aberrant centrioles. Our data help explain the origin of structurally aberrant centrioles and why centriole numerical and structural defects coexist in tumors., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae . The ts- proteins sen2-1HA ts and sam35-2HA ts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HA ts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HA ts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways., Competing Interests: MM, JS, MD, JL, AW No competing interests declared

Multiple organizations have urged a paradigm shift from traditional, whole animal, chemical safety testing to alternative methods. Although these forward-looking methods exist for risk assessment and predication, animal testing is still the preferred method and will remain so until more robust cellular and computational methods are established. To meet this need, we aimed to develop a new, cell division-focused approach based on the idea that defective cell division may be a better predictor of risk than traditional measurements. To develop such an approach, we investigated the toxicity of silver nanoparticles (AgNPs) on human epithelial cells. AgNPs are the type of nanoparticle most widely employed in consumer and medical products, yet toxicity reports are still confounding. Cells were exposed to a range of AgNP doses for both short- and-long term exposure times. The analysis of treated cell populations identified an effect on cell division and the emergence of abnormal nuclear morphologies, including micronuclei and binucleated cells. Overall, our results indicate that AgNPs impair cell division, not only further confirming toxicity to human cells, but also highlighting the propagation of adverse phenotypes within the cell population. Furthermore, this work illustrates that cell division-based analysis will be an important addition to future toxicology studies.

Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells., Competing Interests: RN, DK, KS, TS, SB, JL, MM No competing interests declared, (© 2019, Nanjundappa et al.)

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole's distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.

The centrosome is an important microtubule-organising centre (MTOC) in animal cells. It consists of two barrel-shaped structures, the centrioles, surrounded by the pericentriolar material (PCM), which nucleates microtubules. Centrosomes can form close to an existing structure (canonical duplication) or de novo How centrosomes form de novo is not known. The master driver of centrosome biogenesis, PLK4, is critical for the recruitment of several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where PLK4 can induce de novo MTOC formation ( Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro , PLK4 can self-assemble into condensates that recruit α- and β-tubulins. In Xenopus extracts, PLK4 assemblies additionally recruit STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, forming acentriolar MTOCs de novo The assembly of these robust microtubule asters is independent of dynein, similar to what is found for centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organising catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

In the original version of this Article, the affiliation details for Jadranka Loncarek and Vito Mennella were incorrectly given as 'Cell Biology Program, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, ON, M5G 1X8, Canada' and 'Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, 1050 Boyles Street, Frederick, MD, 21702, USA', respectively. This has now been corrected in both the PDF and HTML versions of the Article.

The inheritance of the centrosome during human fertilization remains mysterious. Here we show that the sperm centrosome contains, in addition to the known typical barrel-shaped centriole (the proximal centriole, PC), a surrounding matrix (pericentriolar material, PCM), and an atypical centriole (distal centriole, DC) composed of splayed microtubules surrounding previously undescribed rods of centriole luminal proteins. The sperm centrosome is remodeled by both reduction and enrichment of specific proteins and the formation of these rods during spermatogenesis. In vivo and in vitro investigations show that the flagellum-attached, atypical DC is capable of recruiting PCM, forming a daughter centriole, and localizing to the spindle pole during mitosis. Altogether, we show that the DC is compositionally and structurally remodeled into an atypical centriole, which functions as the zygote's second centriole. These findings now provide novel avenues for diagnostics and therapeutic strategies for male infertility, and insights into early embryo developmental defects.

The most widely used cancer animal model is the human-murine tumor xenograft. Unbiased molecular dissection of tumor parenchyma versus stroma in human-murine xenografts is critical for elucidating dysregulated protein networks/pathways and developing therapeutics that may target these two functionally codependent compartments. Although antibody-reliant technologies (e.g., immunohistochemistry, imaging mass cytometry) are capable of distinguishing tumor-proper versus stromal proteins, the breadth or extent of targets is limited. Here, we report an antibody-free targeted cross-species glycoproteomic (TCSG) approach that enables direct dissection of human tumor parenchyma from murine tumor stroma at the molecular/protein level in tumor xenografts at a selectivity rate presently unattainable by other means. This approach was used to segment/dissect and obtain the protein complement phenotype of the tumor stroma and parenchyma of the metastatic human lung adenocarcinoma A549 xenograft, with no need for tissue microdissection prior to mass-spectrometry analysis. An extensive molecular map of the tumor proper and the associated microenvironment was generated along with the top functional N-glycosylated protein networks enriched in each compartment. Importantly, immunohistochemistry-based cross-validation of selected parenchymal and stromal targets applied on human tissue samples of lung adenocarcinoma and normal adjacent tissue is indicative of a noteworthy translational capacity for this unique approach that may facilitate identifications of novel targets for next generation antibody therapies and development of real time preclinical tumor models., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest.

The centriole is a multifunctional structure that organizes centrosomes and cilia and is important for cell signaling, cell cycle progression, polarity, and motility. Defects in centriole number and structure are associated with human diseases including cancer and ciliopathies. Discovery of the centriole dates back to the 19th century. However, recent advances in genetic and biochemical tools, development of high-resolution microscopy, and identification of centriole components have accelerated our understanding of its assembly, function, evolution, and its role in human disease. The centriole is an evolutionarily conserved structure built from highly conserved proteins and is present in all branches of the eukaryotic tree of life. However, centriole number, size, and organization varies among different organisms and even cell types within a single organism, reflecting its cell type-specialized functions. In this review, we provide an overview of our current understanding of centriole biogenesis and how variations around the same theme generate alternatives for centriole formation and function., (© 2018 Loncarek and Bettencourt Dias.)

Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

Far-red cyanine fluorophores find extensive use in modern microscopy despite modest quantum yields. To improve the photon output of these molecules, we report a synthetic strategy that blocks the major deactivation pathway: excited-state trans-to-cis polyene rotation. In the key transformation, a protected dialdehyde precursor undergoes a cascade reaction to install the requisite tetracyclic ring system. The resulting molecules exhibit the characteristic features of conformational restraint, including improved fluorescence quantum yield and extended lifetime. Moreover, these compounds recover from hydride reduction with dramatically improved efficiency. These observations enable efficient single-molecule localization microscopy in oxygenated buffer without addition of thiols. Enabled by modern organic synthesis, these studies provide a new class of far-red dyes with promising spectroscopic and chemical properties.

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

The first cell of an animal (zygote) requires centrosomes that are assembled from paternally inherited centrioles and maternally inherited pericentriolar material (PCM) [1]. In some animals, sperm centrioles with typical ultrastructure are the origin of the first centrosomes in the zygote [2-4]. In other animals, however, sperm centrioles lose their proteins and are thought to be degenerated and non-functional during spermiogenesis [5, 6]. Here, we show that the two sperm centrioles (the giant centriole [GC] and the proximal centriole-like structure [PCL]) in Drosophila melanogaster are remodeled during spermiogenesis through protein enrichment and ultrastructure modification in parallel to previously described centrosomal reduction [7]. We found that the ultrastructure of the matured sperm (spermatozoa) centrioles is modified dramatically and that the PCL does not resemble a typical centriole. We also describe a new phenomenon of Poc1 enrichment of the atypical centrioles in the spermatozoa. Using various mutants, protein expression during spermiogenesis, and RNAi knockdown of paternal Poc1, we found that paternal Poc1 enrichment is essential for the formation of centrioles during spermiogenesis and for the formation of centrosomes after fertilization in the zygote. Altogether, these findings demonstrate that the sperm centrioles are remodeled both in their protein composition and in ultrastructure, yet they are functional and are essential for normal embryogenesis in Drosophila., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The breast cancer gene, BRCA2, is essential for viability, yet patients with Fanconi anemia-D1 subtype are born alive with biallelic mutations in this gene. The hypomorphic nature of the mutations is believed to support viability, but this is not always apparent. One such mutation is IVS7+2T>G, which causes premature protein truncation due to skipping of exon 7. We previously identified a transcript lacking exons 4-7, which restores the open-reading frame, encodes a DNA repair proficient protein and is expressed in IVS7+2T>G carriers. However, because the exons 4-7 encoded region contains several residues required for normal cell-cycle regulation and cytokinesis, this transcript's ability to support viability can be argued. To address this, we generated a Brca2 knock-in mouse model lacking exons 4-7 and demonstrated that these exons are dispensable for viability as well as tumor-free survival. This study provides the first in vivo evidence of the functional significance of a minor transcript of BRCA2 that can play a major role in the survival of humans who are homozygous for a clearly pathogenic mutation. Our results highlight the importance of assessing protein function restoration by premature truncating codon bypass by alternative splicing when evaluating the functional significance of variants such as nonsense and frame-shift mutations that are assumed to be clearly pathogenic. Our findings will impact not only the assessment of variants that map to this region, but also influence counseling paradigms and treatment options for such mutation carriers., (Published by Oxford University Press 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Mouse double-minute 1 (Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding., (© 2015 Van de Mark et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Centrosome overduplication promotes mitotic abnormalities, invasion and tumorigenesis. Cells regulate the number of centrosomes by limiting centriole duplication to once per cell cycle. The orthogonal orientation between a mother and a daughter centriole, established at the time of centriole duplication, is thought to block further duplication of the mother centriole. Loss of orthogonal orientation (disengagement) between two centrioles during anaphase is considered a licensing event for the next round of centriole duplication. Disengagement requires the activity of Polo-like kinase 1 (Plk1), but how Plk1 drives this process is not clear. Here we employ correlative live/electron microscopy and demonstrate that Plk1 induces maturation and distancing of the daughter centriole, allowing reduplication of the mother centriole even if the original daughter centriole is still orthogonal to it. We find that mother centrioles can undergo reduplication when original daughter centrioles are only ∼80 nm apart, which is the distance centrioles normally reach during prophase.

In the early mouse embryo, a specialized population of extraembryonic visceral endoderm (VE) cells called the distal VE (DVE) arises at the tip of the egg cylinder stage embryo and then asymmetrically migrates to the prospective anterior, recruiting additional distal cells. Upon migration these cells, called the anterior VE (AVE), establish the anterior posterior (AP) axis by restricting gastrulation-inducing signals to the opposite pole. The Nodal-signaling pathway has been shown to have a critical role in the generation and migration of the DVE/AVE. The Nodal gene is expressed in both the VE and in the pluripotent epiblast, which gives rise to the germ layers. Previous findings have provided conflicting evidence as to the relative importance of Nodal signaling from the epiblast vs. VE for AP patterning. Here we show that conditional mutagenesis of the Nodal gene specifically within the VE leads to reduced Nodal expression levels in the epiblast and incomplete or failed DVE/AVE migration. These results support a required role for VE Nodal to maintain normal levels of expression in the epiblast, and suggest signaling from both VE and epiblast is important for DVE/AVE migration., (Published by Elsevier Inc.)

Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Background: Human PAX-Interacting Protein 1 (PAXIP1)-associated glutamate rich protein 1 (PAGR1, also known as PA1) originally was discovered as part of a complex containing PAXIP1 and histone H3K4 methyltransferases MLL3 and MLL4, suggesting a role in epigenetic gene regulation. Further in vitro studies suggested additional functions in DNA damage repair and transcription. However, in vivo analysis of PAGR1 function has been lacking., Results: Here we show that expression of the cognate mouse gene Pagr1a is found predominately in the extraembryonic and chorionic ectoderm from pregastrulation stages and is up-regulated within the embryo proper after gastrulation. Embryos with a germ line deletion of Pagr1a establish the anterior-posterior axis, and show normal neuroectodermal, mesodermal, and endodermal patterning, but fail to develop beyond the four- to five-somite stage or to undergo axial rotation. Pagr1a(-) (/) (-) embryos also show abnormal development of extraembryonic tissues with defects seen in the amnion, chorion and visceral yolk sac. At the molecular level, Pagr1a(-) (/) (-) embryos have reduced expression of BMP2, a known regulator of extraembryonic development., Conclusions: Loss of mouse Pagr1a function leads to defective extraembryonic development, likely due at least in part to altered BMP signaling, contributing to developmental arrest., (© 2014 Wiley Periodicals, Inc.)

Genome-wide association studies (GWAS) of 10 different cancers have identified pleiotropic cancer predisposition loci across a region of chromosome 5p15.33 that includes the TERT and CLPTM1L genes. Of these, susceptibility alleles for pancreatic cancer have mapped to the CLPTM1L gene, thus prompting an investigation of the function of CLPTM1L in the pancreas. Immunofluorescence analysis indicated that CLPTM1L localized to the endoplasmic reticulum where it is likely embedded in the membrane, in accord with multiple predicted transmembrane domains. Overexpression of CLPTM1L enhanced growth of pancreatic cancer cells in vitro (1.3-1.5-fold; PDAY7 < 0.003) and in vivo (3.46-fold; PDAY68 = 0.039), suggesting a role in tumor growth; this effect was abrogated by deletion of two hydrophilic domains. Affinity purification followed by mass spectrometry identified an interaction between CLPTM1L and non-muscle myosin II (NMM-II), a protein involved in maintaining cell shape, migration, and cytokinesis. The two proteins colocalized in the cytoplasm and, after treatment with a DNA-damaging agent, at the centrosomes. Overexpression of CLPTM1L and depletion of NMM-II induced aneuploidy, indicating that CLPTM1L may interfere with normal NMM-II function in regulating cytokinesis. Immunohistochemical analysis revealed enhanced staining of CLPTM1L in human pancreatic ductal adenocarcinoma (n = 378) as compared with normal pancreatic tissue samples (n = 17; P = 1.7 × 10(-4)). Our results suggest that CLPTM1L functions as a growth-promoting gene in the pancreas and that overexpression may lead to an abrogation of normal cytokinesis, indicating that it should be considered as a plausible candidate gene that could explain the effect of pancreatic cancer susceptibility alleles on chr5p15.33., (©2014 American Association for Cancer Research.)

Centrosomes play an important role in various cellular processes, including spindle formation and chromosome segregation. They are composed of two orthogonally arranged centrioles, whose duplication occurs only once per cell cycle. Accurate control of centriole numbers is essential for the maintenance of genomic integrity. Although it is well appreciated that polo-like kinase 4 (Plk4) plays a central role in centriole biogenesis, how it is recruited to centrosomes and whether this step is necessary for centriole biogenesis remain largely elusive. Here we showed that Plk4 localizes to distinct subcentrosomal regions in a temporally and spatially regulated manner, and that Cep192 and Cep152 serve as two distinct scaffolds that recruit Plk4 to centrosomes in a hierarchical order. Interestingly, Cep192 and Cep152 competitively interacted with the cryptic polo box of Plk4 through their homologous N-terminal sequences containing acidic-α-helix and N/Q-rich motifs. Consistent with these observations, the expression of either one of these N-terminal fragments was sufficient to delocalize Plk4 from centrosomes. Furthermore, loss of the Cep192- or Cep152-dependent interaction with Plk4 resulted in impaired centriole duplication that led to delayed cell proliferation. Thus, the spatiotemporal regulation of Plk4 localization by two hierarchical scaffolds, Cep192 and Cep152, is critical for centriole biogenesis.

Assembly of an integral Golgi complex is driven by microtubule (MT)-dependent transport. Conversely, the Golgi itself functions as an unconventional MT-organizing center (MTOC). This raises the question of whether Golgi assembly requires centrosomal MTs or can be self-organized, relying on its own MTOC activity. The computational model presented here predicts that each MT population is capable of gathering Golgi stacks but not of establishing Golgi complex integrity or polarity. In contrast, the concerted effort of two MT populations would assemble an integral, polarized Golgi complex. Indeed, while laser ablation of the centrosome did not alter already-formed Golgi complexes, acentrosomal cells fail to reassemble an integral complex upon nocodazole washout. Moreover, polarity of post-Golgi trafficking was compromised under these conditions, leading to strong deficiency in polarized cell migration. Our data indicate that centrosomal MTs complement Golgi self-organization for proper Golgi assembly and motile-cell polarization.

Supernumerary centrioles lead to abnormal mitosis, which in turn promotes tumorigenesis. Thus, centriole duplication must be coordinated with the cell cycle to ensure that the number of centrioles in the cell doubles precisely during each cell cycle. However, in some transformed cells, centrioles undergo multiple rounds of duplication (reduplication) during prolonged interphase. Mechanisms responsible for centriole reduplication are poorly understood. Here, we report that centrioles reduplicate consistently in cancerous and nontransformed human cells during G2 arrests and that this reduplication requires the activity of Polo-like kinase 1 (Plk1). We also find that a cell's ability to reduplicate centrioles during S arrests depends on the presence of activated (Thr210-phosphorylated) Plk1 at the centrosome. In the absence of activated Plk1, nascent procentrioles remain associated with mother centrioles, which prevents centriole reduplication. In contrast, if Plk1(pT210) appears at the centrosome, procentrioles mature, disengage from mother centrioles, and ultimately duplicate. Plk1 activity is not required for the assembly of procentrioles, however. Thus, the role of Plk1 is to coordinate the centriole duplication cycle with the cell cycle. Activation of Plk1 during late S/G2 induces procentriole maturation, and after this point, the centriole cycle can be completed autonomously, even in the absence of cell-cycle progression., (2010 Elsevier Ltd. All rights reserved.)

During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.

The centrosome is the principal microtubule organizing center (MTOC) of animal cells. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division.

The centrosome, an organelle comprising centrioles and associated pericentriolar material, is the major microtubule organizing center in animal cells. For the cell to form a bipolar mitotic spindle and ensure proper chromosome segregation at the end of each cell cycle, it is paramount that the cell contains two and only two centrosomes. Because the number of centrosomes in the cell is determined by the number of centrioles, cells have evolved elaborate mechanisms to control centriole biogenesis and to tightly coordinate this process with DNA replication. Here we review key proteins involved in centriole assembly, compare two major modes of centriole biogenesis, and discuss the mechanisms that ensure stringency of centriole number.

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.