Neuronal non-CG methylation is an essential target for MeCP2 function.

DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

Convergent genes shape budding yeast pericentromeres.

The three-dimensional architecture of the genome governs its maintenance, expression and transmission. The cohesin protein complex organizes the genome by topologically linking distant loci, and is highly enriched in specialized chromosomal domains surrounding centromeres, called pericentromeres1-6. Here we report the three-dimensional structure of pericentromeres in budding yeast (Saccharomyces cerevisiae) and establish the relationship between genome organization and function. We find that convergent genes mark pericentromere borders and, together with core centromeres, define their structure and function by positioning cohesin. Centromeres load cohesin, and convergent genes at pericentromere borders trap it. Each side of the pericentromere is organized into a looped conformation, with border convergent genes at the base. Microtubule attachment extends a single pericentromere loop, size-limited by convergent genes at its borders. Reorienting genes at borders into a tandem configuration repositions cohesin, enlarges the pericentromere and impairs chromosome biorientation during mitosis. Thus, the linear arrangement of transcriptional units together with targeted cohesin loading shapes pericentromeres into a structure that is competent for chromosome segregation. Our results reveal the architecture of the chromosomal region within which kinetochores are embedded, as well as the restructuring caused by microtubule attachment. Furthermore, we establish a direct, causal relationship between the three-dimensional genome organization of a specific chromosomal domain and cellular function.

Tissue-Specific Gene Repositioning by Muscle Nuclear Membrane Proteins Enhances Repression of Critical Developmental Genes during Myogenesis.

Whether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes ⅓ to ⅔ of a gene's normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation.

Microtubule-independent movement of the fission yeast nucleus.

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. The vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum-plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells.

Aerosol and bioaerosol particle size and dynamics from defective sanitary plumbing systems.

Aerosols are readily transported on airstreams through building sanitary plumbing and sewer systems, and those containing microbial pathogens (known as bioaerosols) are recognized as contributors to infection spread within buildings. When a defect occurs in the sanitary plumbing system that affects the system integrity, a cross-transmission route is created that can enable the emission of bioaerosols from the system into the building. These emission occurrences are characterized as short-burst events (typically <1 min in duration) which make them difficult to detect and predict. The characterization of these emission events is the focus of this research. Two methods were used to characterize bioaerosol emission events in a full-scale test rig: (a) an Aerodynamic Particle Sizer (APS) for particle size distribution and concentrations; and (b) a slit-to-agar sampler to enumerate the ingress of a viable tracer microorganism (Pseudomonas putida). The APS data confirmed that most particles (>99.5%) were <5 µm and were therefore considered aerosols. Particles generated within the sanitary plumbing system as a result of a toilet flush leads to emissions into the building during system defect conditions with an equivalence of someone talking loudly for over 6 and a half minutes. There were no particles detected of a size >11 µm anywhere in the system. Particle count was influenced by toilet flush volume, but it was not possible to determine if there was any direct influence from airflow rate since both particle and biological data showed no correlation with upward airflow rates and velocities. Typical emissions resulting from a 6 L toilet flush were in the range of 280-400 particles per second at a concentration of typically 9-12 number per cm3 and a total particle count in the region of 3000 to 4000 particles, whereas the peak emissions from a 1.2 L toilet flush were 60-80 particles per second at a concentration of 2.4-3 number per cm3 and a total particle count in the region of 886 to 1045 particles. The reduction in particles is in direct proportion to the reduction in toilet flush volume. The slit-to-agar sampler was able to provide viable time course CFU data and confirmed the origin of the particles to be the tracer microorganism flushed into the system. The time course data also have characteristics consistent with the unsteady nature of a toilet flush.

Major Lower Limb Amputation: Outcomes are Improving.

BACKGROUND: Outcomes following major lower limb amputation (MLLA) between 2000 and 2002 from the Department of Vascular Surgery at Royal Perth Hospital have been published; mean postoperative length of stay 20 days, inpatient complication rate 54%, and 30-day mortality 10%. The last decade has seen increasing endovascular revascularization techniques, increased focus on MLLA patients, and general improvements in the model of care. The aim of this study is to compare outcomes between 2000-2002 and 2010-2012. METHODS: Data on all patients undergoing MLLA, transtibial or proximal, in the 2 time periods were extracted from the department of vascular surgery database. Medical records, government registries, and phone calls to primary care providers were used to clarify mortality. RESULTS: Limb ischemia remains the most common indication for MLLA with smoking, hypertension, and diabetes being the main comorbid diseases. The rates of wound infections have fallen from 26.4% to 12.4% (P = 0.023), rate of admission to ICU has fallen from 48.3% to 17.5% (P = 0.001), and revision amputation to a higher level has fallen from 11.5% to 7.2% (P = 0.043). Acute hospital, postoperative length of stay has trended down from 15.74 to 20.29 days (P = 0.075). Mortality overall has fallen from 60.92% to 46.39% (P = 0.049). Thirty-day mortality fallen from 10.34% to 5.15% (P = 0.185), 6-month 28.76% to 16.5% (P = 0.046), and 1-year 40.22% to 21.65% (P = 0.006). CONCLUSIONS: Patients undergoing MLLA still carry a high burden of comorbid disease. With changes in revascularization technique, consultant supervision, and multidisciplinary model of care, we have seen the rate of complications fall, length of stay trend down, and overall mortality reduce. Despite improvements, outcomes remain sobering and more can be done.

Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochore.

Kinetochores assemble on distinct 'centrochromatin' containing the histone H3 variant CENP-A and interspersed nucleosomes dimethylated on H3K4 (H3K4me2). Little is known about how the chromatin environment at active centromeres governs centromeric structure and function. Here, we report that centrochromatin resembles K4-K36 domains found in the body of some actively transcribed housekeeping genes. By tethering the lysine-specific demethylase 1 (LSD1), we specifically depleted H3K4me2, a modification thought to have a role in transcriptional memory, from the kinetochore of a synthetic human artificial chromosome (HAC). H3K4me2 depletion caused kinetochores to suffer a rapid loss of transcription of the underlying α-satellite DNA and to no longer efficiently recruit HJURP, the CENP-A chaperone. Kinetochores depleted of H3K4me2 remained functional in the short term, but were defective in incorporation of CENP-A, and were gradually inactivated. Our data provide a functional link between the centromeric chromatin, α-satellite transcription, maintenance of CENP-A levels and kinetochore stability.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function.

Human kinetochores are transcriptionally active, producing very low levels of transcripts of the underlying alpha-satellite DNA. However, it is not known whether kinetochores can tolerate acetylated chromatin and the levels of transcription that are characteristic of housekeeping genes, or whether kinetochore-associated 'centrochromatin', despite being transcribed at a low level, is essentially a form of repressive chromatin. Here, we have engineered two types of acetylated chromatin within the centromere of a synthetic human artificial chromosome. Tethering a minimal NF-κB p65 activation domain within kinetochore-associated chromatin produced chromatin with high levels of histone H3 acetylated on lysine 9 (H3K9ac) and an ~10-fold elevation in transcript levels, but had no substantial effect on kinetochore assembly or function. By contrast, tethering the herpes virus VP16 activation domain produced similar modifications in the chromatin but resulted in an ~150-fold elevation in transcripts, approaching the level of transcription of an endogenous housekeeping gene. This rapidly inactivated kinetochores, causing a loss of assembled CENP-A and blocking further CENP-A assembly. Our data reveal that functional centromeres in vivo show a remarkable plasticity--kinetochores tolerate profound changes to their chromatin environment, but appear to be critically sensitive to the level of centromeric transcription.

Impact of intraoperative vasopressor use in free tissue transfer for head, neck, and extremity reconstruction.

General anesthesia induces hypotension and this is commonly treated intraoperatively with administering vasopressors. Microsurgeons are hesitant to use vasopressors due to the potential risk of inducing vasoconstriction and flap necrosis. The aim of this study was to determine the frequency of intraoperative vasopressor utilization in patients undergoing free tissue transfer reconstruction and to determine its impact on patient outcomes. An IRB-approved retrospective review was performed for 47 consecutive patients undergoing free tissue transfer for head, neck, and extremity reconstruction at Wake Forest Baptist Health over a 3-year period. Free flap survival was 97%, with 3% of patients having total flap necrosis and 17% with partial flap necrosis. The frequency of intraoperative vasopressor use was 53.2%. There was no significant difference in the frequency of total or partial flap necrosis between patients who received intraoperative vasopressors and those who did not. Similarly, there was no statistical significance in the rate of arterial or venous thrombosis between the 2 groups (P = 0.095 and P = 0.095, respectively). The use of vasopressors did not significantly increase postoperative complications. The timing of vasopressor administration did not affect outcomes. Intraoperative vasopressors are used more frequently than previously realized during free tissue transfer for reconstructive surgery. The use of intraoperative vasopressors does not appear to adversely affect outcomes of free tissue transfer. Further investigation and larger study size are needed to analyze the timing of drug administration, dose, and type of vasopressor to better understand the impact of intraoperative vasopressor use in free tissue transfer outcomes.

Aesthetic outcomes of acellular dermal matrix in tissue expander/implant-based breast reconstruction.

INTRODUCTION: Tissue expander and implant-based breast reconstruction after mastectomy is the most common method of breast reconstruction. Modifications of the traditional total submuscular reconstruction (TSR) have been made using acellular dermal matrix (ADM) to create an inferolateral sling and a more natural implant pocket for superior aesthetic results. The objective of this study was to assess aesthetic outcomes when using ADM in breast reconstruction. METHODS: A retrospective chart review identified all patients who underwent implant-based breast reconstruction from 2005 to 2009 at our institution. Demographic information, complications, reoperations, and aesthetic outcome data were collected for all patients meeting inclusion criteria related to adequate follow-up and postoperative photographs. Five aesthetic outcomes were evaluated for all study patients by 18 blinded evaluators using postoperative photographs. Outcomes were scored on a scale of 1 to 5, with 5 representing the best possible aesthetic score. RESULTS: A total of 122 patients underwent 183 tissue expander-based reconstructions (ADM, n = 58; TSR, n = 125). The infection rate in patients with ADM was 16.2% compared to 5.9% in TSR patients, but this was not statistically significant (P = 0.09). Capsular contracture was more common in TSR patients (23.5%), compared to those with ADM (8.1%), P = 0.048. Aesthetic scores from the attending plastic surgeons were as follows: natural contour (ADM, 3.36; TSR, 3.02; P = 0.0001), symmetry of shape (ADM, 3.57; TSR, 3.27; P = 0.005), symmetry of size (ADM, 3.68; TSR, 3.42; P = 0.002), position on chest wall (ADM, 3.75; TSR, 3.45; P = 0.004), and overall aesthetic appearance (ADM, 3.56; TSR, 3.20; P = 0.0001). CONCLUSIONS: For all 5 aesthetic parameters evaluated, the ADM group scored significantly higher than the TSR group by 18 blinded evaluators. These consistent findings suggest that the use of ADM in breast reconstruction does confer a significant advantage in aesthetic outcomes for breast reconstruction. This is likely at the cost of a higher infection rate when using ADM; however, that may be offset by the advantage of a lower rate of capsular contracture in patients with ADM.

Rapid response of thyroid eye disease, peripheral edema, and acropathy to teprotumumab infusion.

Purpose: We present a case of rapid improvement in symptoms of thyroid eye disease and amelioration of worsening peripheral edema and acropathy with infusion of teprotumumab, a monoclonal antibody targeting the insulin-like growth factor-1 receptor. Observations: A 66 year old female with history of Hashimoto thyroiditis developed progressive thyroid eye disease (TED), peripheral edema, and acropathy attributable to acute Graves disease. Her signs and symptoms, refractory to oral steroid and diuretic therapy, rapidly improved following a standard dosing regimen of teprotumumab (one infusion 10 mg/kg then seven infusions 20 mg/kg) to resolution. Conclusions & importance: Teprotumumab, a monoclonal antibody targeting the insulin-like growth factor-1 receptor, is the first medication approved by the FDA for use in TED. Teprotumumab may contribute to the treatment of extraocular manifestations of Graves disease, chief among these peripheral soft tissue manifestations.

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation.

Correlative evidence has suggested that the methyl-CpG-binding protein MeCP2 contributes to the formation of heterochromatin condensates via liquid-liquid phase separation. This interpretation has been reinforced by the observation that heterochromatin, DNA methylation and MeCP2 co-localise within prominent foci in mouse cells. The findings presented here revise this view. MeCP2 localisation is independent of heterochromatin as MeCP2 foci persist even when heterochromatin organisation is disrupted. Additionally, MeCP2 foci fail to show hallmarks of phase separation in live cells. Importantly, we find that mouse cellular models are highly atypical as MeCP2 distribution is diffuse in most mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which is a mouse subspecies lacking methylated satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form condensates and its localisation is independent of heterochromatin. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns.

Age-dependent loss of cohesion protection in human oocytes.

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Outcome analysis of 541 women undergoing breast conservation therapy.

Breast conservation therapy (BCT) has evolved as a favorable approach to the management of early-stage breast cancer. Shortcomings of BCT include the potential need for re-excision in the event of positive tumor margins as well as the untoward sequelae of radiation therapy. Both of those factors have led to a substantial proportion of patients undergoing BCT who ultimately report suboptimal aesthetic outcomes. Application of plastic surgery principles to the management of this patient subset has been shown to be beneficial from both an oncologic and cosmetic perspective.The aim of this study was to identify factors that may predict which patients would benefit most from involvement of a plastic surgeon before BCT. A retrospective analysis was performed on 762 patients undergoing lumpectomy during a 10-year study period at a single institution. Younger women and patients with tumor size approaching 2 cm were noted to have a significantly higher likelihood of oncologic outcomes that ultimately required breast reconstruction. Integration of oncoplastic techniques in the surgical management of patients undergoing BCT would likely contribute to improvement in aesthetic outcomes and overall patient satisfaction.

AlloDerm revision for failed pharyngoplasty.

Velopharyngeal insufficiency (VPI) occurs in more than 20% of patients with a cleft palate after primary palatoplasty. Surgical treatment focuses on pharyngoplasty to narrow the nasopharyngeal space and to decrease the distance needed for palatal closure. Persistent VPI after pharyngoplasty affects more than 20% of patients.From September 2007 to December 2009, 16 children (10 boys and 6 girls) with a mean age of 9.5 years (4-15 years) underwent surgical revision using an AlloDerm sling for persistent VPI after at least 1 previous failed pharyngoplasty. Ten children had previous sphincter pharyngoplasties, and 6 had previous pharyngeal flaps. Surgical technique involves creation of a submucosal tunnel through the limbs of the previous pharyngoplasty or pharyngeal flap. A strip of AlloDerm is threaded circumferentially, and the port is adjusted to the desired aperture.All patients underwent preoperative and postoperative analysis of VPI, including oral pharyngeal and perceptual speech examination by speech pathology with a mean follow-up of 441 days. Acoustic nasometry was used to objectively compare preoperative and postoperative nasalance values. A significant improvement in perceptual resonance was seen in 93.8% of patients, and 87.5% of patients improved to normal or mild resonance (P < 0.001). There was a significant mean reduction of nasalance using the MacKay-Kummer Simplified Nasometric Assessment Procedure test (P < 0.001). Two patients developed postoperative flap dehiscence, with one being revised ultimately to have normal speech resonance.Revision pharyngoplasty using an AlloDerm sling can safely and effectively improve speech in patients with persistent VPI after failed pharyngoplasty. Long-term follow-up studies are ongoing.

Establishment of centromere identity is dependent on nuclear spatial organization.

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

Mechanistic basis for Sgo1-mediated centromere localization and function of the CPC.

Centromere association of the chromosomal passenger complex (CPC; Borealin-Survivin-INCENP-Aurora B) and Sgo1 is crucial for chromosome biorientation, a process essential for error-free chromosome segregation. Phosphorylated histone H3 Thr3 (H3T3ph; directly recognized by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognized via Sgo1), together with CPC's intrinsic nucleosome-binding ability, facilitate CPC centromere recruitment. However, the molecular basis for CPC-Sgo1 binding and how their physical interaction influences CPC centromere localization are lacking. Here, using an integrative structure-function approach, we show that the "histone H3-like" Sgo1 N-terminal tail-Survivin BIR domain interaction acts as a hotspot essential for CPC-Sgo1 assembly, while downstream Sgo1 residues and Borealin contribute for high-affinity binding. Disrupting Sgo1-Survivin interaction abolished CPC-Sgo1 assembly and perturbed CPC centromere localization and function. Our findings reveal that Sgo1 and H3T3ph use the same surface on Survivin to bind CPC. Hence, it is likely that these interactions take place in a spatiotemporally restricted manner, providing a rationale for the Sgo1-mediated "kinetochore-proximal" CPC centromere pool.

Activated alleles of the Schizosaccharomyces pombe gpa2+ Galpha gene identify residues involved in GDP-GTP exchange.

The Schizosaccharomyces pombe glucose/cyclic AMP (cAMP) signaling pathway includes the Gpa2-Git5-Git11 heterotrimeric G protein, whose Gpa2 Galpha subunit directly binds to and activates adenylate cyclase in response to signaling from the Git3 G protein-coupled receptor. To study intrinsic and extrinsic regulation of Gpa2, we developed a plasmid-based screen to identify mutationally activated gpa2 alleles that bypass the loss of the Git5-Git11 Gbetagamma dimer to repress transcription of the glucose-regulated fbp1(+) gene. Fifteen independently isolated mutations alter 11 different Gpa2 residues, with all but one conferring a receptor-independent activated phenotype upon integration into the gpa2(+) chromosomal locus. Biochemical characterization of three activated Gpa2 proteins demonstrated an increased GDP-GTP exchange rate that would explain the mechanism of activation. Interestingly, the amino acid altered in the Gpa2(V90A) exchange rate mutant protein is in a region of Gpa2 with no obvious role in Galpha function, thus extending our understanding of Galpha protein structure-function relationships.

The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly.

Kinetochores direct chromosome segregation in mitosis and meiosis. Faithful gamete formation through meiosis requires that kinetochores take on new functions that impact homolog pairing, recombination, and the orientation of kinetochore attachment to microtubules in meiosis I. Using an unbiased proteomics pipeline, we determined the composition of centromeric chromatin and kinetochores at distinct cell-cycle stages, revealing extensive reorganization of kinetochores during meiosis. The data uncover a network of meiotic chromosome axis and recombination proteins that bind to centromeres in the absence of the microtubule-binding outer kinetochore sub-complexes during meiotic prophase. We show that the Ctf19cCCAN inner kinetochore complex is essential for kinetochore organization in meiosis. Our functional analyses identify a Ctf19cCCAN-dependent kinetochore assembly pathway that is dispensable for mitotic growth but becomes critical upon meiotic entry. Therefore, changes in kinetochore composition and a distinct assembly pathway specialize meiotic kinetochores for successful gametogenesis.