The spatial arrangement of chromosomes during prometaphase facilitates spindle assembly.

Error-free chromosome segregation requires stable attachment of sister kinetochores to the opposite spindle poles (amphitelic attachment). Exactly how amphitelic attachments are achieved during spindle assembly remains elusive. We employed photoactivatable GFP and high-resolution live-cell confocal microscopy to visualize complete 3D movements of individual kinetochores throughout mitosis in nontransformed human cells. Combined with electron microscopy, molecular perturbations, and immunofluorescence analyses, this approach reveals unexpected details of chromosome behavior. Our data demonstrate that unstable lateral interactions between kinetochores and microtubules dominate during early prometaphase. These transient interactions lead to the reproducible arrangement of chromosomes in an equatorial ring on the surface of the nascent spindle. A computational model predicts that this toroidal distribution of chromosomes exposes kinetochores to a high density of microtubules which facilitates subsequent formation of amphitelic attachments. Thus, spindle formation involves a previously overlooked stage of chromosome prepositioning which promotes formation of amphitelic attachments.

ANKRD26 recruits PIDD1 to centriolar distal appendages to activate the PIDDosome following centrosome amplification.

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.

Differential sensitivity of the yeast Lon protease Pim1p to impaired mitochondrial respiration.

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.

An engineered cell line with a hRpn1-attached handle to isolate proteasomes.

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.

SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

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.

Human centrosome organization and function in interphase and mitosis.

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.

PLK4 is a microtubule-associated protein that self-assembles promoting de novo MTOC formation.

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 ß-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 ß-tubulins, leading to MTOC formation.This article has an associated First Person interview with the first author of the paper.

BRCA2 minor transcript lacking exons 4-7 supports viability in mice and may account for survival of humans with a pathogenic biallelic mutation.

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.

Cyanine Conformational Restraint in the Far-Red Range.

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.

Nodal signaling from the visceral endoderm is required to maintain Nodal gene expression in the epiblast and drive DVE/AVE migration.

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.

Hierarchical recruitment of Plk4 and regulation of centriole biogenesis by two centrosomal scaffolds, Cep192 and Cep152.

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.

Loss of function of mouse Pax-Interacting Protein 1-associated glutamate rich protein 1a (Pagr1a) leads to reduced Bmp2 expression and defects in chorion and amnion development.

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.

Endogenous EWSR1 Exists in Two Visual Modalities That Reflect Its Associations with Nucleic Acids and Concentration at Sites of Active Transcription.

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.

The centromere geometry essential for keeping mitosis error free is controlled by spindle forces.

Accurate segregation of chromosomes, essential for the stability of the genome, depends on 'bi-orientation'-simultaneous attachment of each individual chromosome to both poles of the mitotic spindle. On bi-oriented chromosomes, kinetochores (macromolecular complexes that attach the chromosome to the spindle) reside on the opposite sides of the chromosome's centromere. In contrast, sister kinetochores shift towards one side of the centromere on 'syntelic' chromosomes that erroneously attach to one spindle pole with both sister kinetochores. Syntelic attachments often arise during spindle assembly and must be corrected to prevent chromosome loss. It is assumed that restoration of proper centromere architecture occurs automatically owing to elastic properties of the centromere. Here we test this assumption by combining laser microsurgery and chemical biology assays in cultured mammalian cells. We find that kinetochores of syntelic chromosomes remain juxtaposed on detachment from spindle microtubules. These findings reveal that correction of syntelic attachments involves an extra step that has previously been overlooked: external forces must be applied to move sister kinetochores to the opposite sides of the centromere. Furthermore, we demonstrate that the shape of the centromere is important for spindle assembly, because bipolar spindles do not form in cells lacking centrosomes when multiple chromosomes with juxtaposed kinetochores are present. Thus, proper architecture of the centromere makes an important contribution to achieving high fidelity of chromosome segregation.

Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning.

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.

EWSR1's visual modalities are defined by its association with nucleic acids and RNA polymerase II.

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.

Abnormal centriolar biomarker ratios correlate with unexplained bull artificial insemination subfertility: a pilot study.

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.

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network.

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.

Cell cycle progression and de novo centriole assembly after centrosomal removal in untransformed human cells.

How centrosome removal or perturbations of centrosomal proteins leads to G1 arrest in untransformed mammalian cells has been a mystery. We use microsurgery and laser ablation to remove the centrosome from two types of normal human cells. First, we find that the cells assemble centrioles de novo after centrosome removal; thus, this phenomenon is not restricted to transformed cells. Second, normal cells can progress through G1 in its entirety without centrioles. Therefore, the centrosome is not a necessary, integral part of the mechanisms that drive the cell cycle through G1 into S phase. Third, we provide evidence that centrosome loss is, functionally, a stress that can act additively with other stresses to arrest cells in G1 in a p38-dependent fashion.

CPAP insufficiency leads to incomplete centrioles that duplicate but fragment.

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.