Dysfunction of Human Estrogen Signaling as a Novel Molecular Signature of Polycystic Ovary Syndrome.

Estradiol (E2) is a major hormone-controlling folliculogenesis whose dysfunction may participate in polycystic ovary syndrome (PCOS) infertility. To determine whether both the concentration and action of E2 could be impaired in non-hyperandrogenic overweight PCOS women, we isolated granulosa cells (GCs) and follicular fluid (FF) from follicles of women undergoing ovarian stimulation (27 with PCOS, and 54 without PCOS). An analysis of the transcript abundance of 16 genes in GCs showed that androgen and progesterone receptor expressions were significantly increased in GCs of PCOS (by 2.7-fold and 1.5-fold, respectively), while those of the steroidogenic enzymes CYP11A1 and HSD3B2 were down-regulated (by 56% and 38%, respectively). Remarkably, treatment of GC cultures with E2 revealed its ineffectiveness in regulating the expression of several key endocrine genes (e.g., GREB1 or BCL2) in PCOS. Additionally, a comparison of the steroid concentrations (measured by GC/MS) in GCs with those in FF of matched follicles demonstrated that the significant decline in the E2 concentration (by 23%) in PCOS FF was not the result of the E2 biosynthesis reduction. Overall, our study provides novel hallmarks of PCOS by highlighting the ineffective E2 signaling in GCs as well as the dysregulation in the expression of genes involved in follicular growth, which may contribute to aberrant folliculogenesis in non-hyperandrogenic women with PCOS.

Dyspnea induced by inspiratory loading limits dual-tasking in healthy young adults.

OBJECTIVES: Dyspnea is a common and multidimensional experience of healthy adults and those with respiratory disorders. Due to its neural processing, it may limit or interfere with cognition, which may be examined with a dual-task paradigm. The aim of this study was to compare single-task performance of Stroop Colour and Word Test (SCWT) or inspiratory threshold loading (ITL) to their combined dual-task performance. Secondly, whether mood was related to dyspnea or cognitive performance was also evaluated. MATERIALS & METHODS: A virtual pre-post design examined single (SCWT and ITL) and dual-task (SCWT+ITL) performance. For ITL, a Threshold Trainer™ was used to elicit a "somewhat severe" rating of dyspnea. The SCWT required participants to indicate whether a colour-word was congruent or incongruent with its semantic meaning. The Depression, Anxiety and Stress Scale-21 (DASS-21) was completed to assess mood. Breathing frequency, Borg dyspnea rating, and breathing endurance time were ascertained. RESULTS: Thirty young healthy adults (15F, 15M; median age = 24, IQR [23-26] years) completed the study. SCWT+ITL had lower SCWT accuracy compared to SCWT alone (98.6%, [97.1-100.0] vs 99.5%, [98.6-100.0]; p = 0.009). Endurance time was not different between ITL and SCWT+ITL (14.5 minutes, [6.9-15.0]) vs 13.7 minutes, [6.1-15.0]; p = 0.59). DASS-21 scores positively correlated with dyspnea scores during ITL (rho = 0.583, p<0.001) and SCWT+ITL (rho = 0.592, p<0.001). CONCLUSIONS: ITL significantly reduced dual-task performance in healthy young adults. Lower mood was associated with greater perceived dyspnea during single and dual-task ITL. Considering the prevalence of dyspnea in respiratory disorders, the findings of this dual task paradigm warrant further exploration to inform dyspnea management during daily activities.

Interventions Including Smart Technology Compared With Face-to-face Physical Activity Interventions in Older Adults: Systematic Review and Meta-analysis.

BACKGROUND: This is a systematic review of randomized controlled trials and a meta-analysis comparing smart technology with face-to-face physical activity (PA) interventions in community-dwelling older adults (mean age 60 years). OBJECTIVE: This study aims to determine the effect of interventions including smart technology components compared with face-to-face PA interventions on PA and physical function in older adults. The secondary outcomes are depression, anxiety, and health-related quality of life. METHODS: We searched MEDLINE, Embase, CINAHL, and AMED electronic databases from inception to February 2021. Two independent reviewers screened titles, abstracts, and full texts and performed data extraction and risk of bias assessments using the Cochrane risk of bias tool. The Grading of Recommendations Assessment, Development and Evaluation was used to evaluate the quality of the evidence. We provided a narrative synthesis on all included studies and, where possible, performed meta-analyses for similar outcomes. RESULTS: This review included 19 studies with a total of 3455 participants. Random effects meta-analyses showed that interventions with smart technology components resulted in improved step count (mean difference 1440 steps, 95% CI 500-2390) and total PA (standardized mean difference 0.17, 95% CI 0.02-0.32) compared with face-to-face alone. There was no difference between groups in terms of the measures of physical function. Smart technology alone did not show significant differences between groups in any outcome. The quality of the evidence was very low based on the Grading of Recommendations Assessment, Development and Evaluation criteria. CONCLUSIONS: Interventions that include smart technology may improve daily step counts by an average of 1440 steps in community-dwelling older adults; however, the quality of the evidence was very low. Future studies are needed to improve the certainty of these results. TRIAL REGISTRATION: PROSPERO CRD42020135232; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=135232.

Estradiol Signaling at the Heart of Folliculogenesis: Its Potential Deregulation in Human Ovarian Pathologies.

Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ERα and ERß. These transcription factors are differentially expressed within follicles, with ERß being the predominant ER in GC. Several ERß splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ERß-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ERß splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Experiences of a home-based fall prevention exercise program among older adults with chronic lung disease.

BACKGROUND: Individuals with chronic obstructive pulmonary disease (COPD) often have mobility limitations; these may include challenges with balance and being at high risk of falling. Risk of falling can be reduced through exercise programs targeting balance; however, older adults with COPD may experience many barriers to exercise adherence. In this paper we present qualitative findings about the feasibility of a six-month home-based fall-prevention exercise program for older adults with COPD. The aim of the study is to describe the experiences of older adults with COPD who participated in a home-based fall prevention exercise program in order to determine their perceived facilitators and barriers to participation. METHODS: 15 participants with COPD who had completed the six-month home-based program participated in one-on-one semi-structured interviews over the phone. Interpretive description methodology and thematic analysis were used. RESULTS: Two major themes emerged with respect to participants' perspectives of the intervention and facilitators and barriers to participation: program personalization based on each individual's characteristics, lifestyles, and preferences; and self-motivation and support from family, friends, and healthcare providers. CONCLUSIONS: Fall prevention exercise programs that are personalized and focus on providing support for older adults with COPD may help to improve adherence and reduce participants' risk of falling.Implications for rehabilitationIndividuals with COPD often have balance problems and a high risk of falling.Fall prevention programs can improve balance, but adherence is a commonly cited challenge.Patient experiences suggest that fall prevention programs should be personalized and incorporate social support to improve adherence to fall prevention exercises.

The Effect of Lifestyle Interventions on the International Classification of Functioning, Disability and Health Participation Domain in Older Adults: A Systematic Review and Meta-Analysis.

BACKGROUND AND OBJECTIVES: The World Health Organization's International Classification of Functioning, Disability and Health (ICF) recognizes participation in life situations as a major component of health. Identifying interventions that target this component is critical, particularly in older adulthood, where declines in physical functioning can impact participation. The purpose of this study was to evaluate the effectiveness of lifestyle or behavior change interventions on the ICF participation domain in older adults. RESEARCH DESIGN AND METHODS: MEDLINE (Ovid), Embase (Ovid), CINAHL (EBSCO), AgeLine (EBSCO), PsycINFO (Ovid), and AMED (Ovid) were searched from inception to April 2020 for randomized controlled trials comparing the effects of any behavior change or lifestyle intervention to usual care among community-dwelling adults ≥60 years with respect to participation-related domains of the ICF. The protocol was registered with Prospero (CRD42019125334). RESULTS: Eight studies with a total of 1,548 participants were included. No significant effect on participation outcomes was found (standardized mean difference 0.04; 95% CI -0.19 to 0.26; p = .76) and the quality of evidence was judged to be very low. DISCUSSION AND IMPLICATIONS: Lifestyle or behavior change interventions showed limited effect on participation in later life. However, there remains much uncertainty in the estimate of this effect due, in part, to the low quality of the included studies. Measurement tools that are responsive to changes in participation in older adulthood should be used to determine the effect of such interventions. Improving study design will lead to more efficacious interventions that promote participation for our aging population.

Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Smart technology vs. face-to-face physical activity interventions in older adults: a systematic review protocol.

OBJECTIVE: The objective of this review is to determine the effect of physical activity interventions delivered via smart technology compared with face-to-face interventions for improving physical activity and physical function in older adults. INTRODUCTION: Physical activity is a modifiable risk factor for multiple noncommunicable diseases and reduces the risk of premature mortality. Despite this, one in four adults does not meet recommended levels of physical activity. This pattern of inactivity increases with age. Smart technology, such as wearables, tablets, or laptops, is one solution for improving physical activity. Research has shown that different smart technology solutions can increase physical activity in older adults. While individual studies support smart technology to increase physical activity, there are no systematic reviews comparing the effects of smart technology with traditional face-to-face physical activity interventions. INCLUSION CRITERIA: We will include randomized controlled trials of physical activity interventions delivered via smart technology (eg, wearables, tablets, computers) compared with face-to-face (ie, in person) interventions for community-dwelling older adults aged 60 years or older. METHODS: We will search four databases (AMED, CINAHL, Embase, MEDLINE) from inception for relevant studies. All abstracts and full texts will be screened independently and in duplicate. Risk of bias, data extraction, and quality assessment will be completed in the same manner. If possible, a meta-analysis will be performed of the primary outcomes of physical activity, physical function, and adherence rate. Subgroup analyses will be conducted by type of physical activity, and type of smart technology, where possible. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020135232.

Estradiol Regulates mRNA Levels of Estrogen Receptor Beta 4 and Beta 5 Isoforms and Modulates Human Granulosa Cell Apoptosis.

Estrogen receptor beta (ERß) plays a critical role in granulosa cell (GC) functions. The existence of four human ERß splice isoforms in the ovary suggests their differential implication in 17ß-estradiol (E2) actions on GC apoptosis causing follicular atresia. In this study, we investigated whether E2 can regulate ERß isoforms expression to fine tune its apoptotic activities in human GC. For this purpose, we measured by RT-qPCR the expression of ERß isoforms in primary culture of human granulosa cells (hGCs) collected from patients undergoing in vitro fertilization, before and after E2 exposure. Besides, we assessed the potential role of ERß isoforms on cell growth and apoptosis after their overexpression in a human GC line (HGrC1 cells). We confirmed that ERß1, ERß2, ERß4, and ERß5 isoform mRNAs were predominant over that of ERα in hGCs, and found that E2 selectively regulates mRNA levels of ERß4 and ERß5 isoforms in these cells. In addition, we demonstrated that overexpression of ERß1 and ERß4 in HGrC1 cells increased cell apoptosis by 225% while ERß5 or ERß2 had no effect. Altogether, our study revealed that E2 may influence GC fate by specifically regulating the relative abundance of ERß isoforms mRNA to modulate the balance between pro-apoptotic and non-apoptotic ERß isoforms.

Which Balance Subcomponents Distinguish Between Fallers and Non-Fallers in People with COPD?

Rationale: Chronic obstructive pulmonary disease (COPD) is an increasingly prevalent lung disease linked to dysfunctional balance and an increased risk of falls. The Balance Evaluation Systems Test (BESTest) evaluates the six underlying subcomponents of functional balance. The aim of this study was to determine the specific balance subcomponents and cut-off scores that discriminate between fallers and non-fallers with COPD to guide fall risk assessment and prevention. Methods: A secondary analysis of cross-sectional data from two prior studies in COPD was performed. Independent samples t-tests were used to explore the differences in the BESTest sub-system scores between fallers and non-fallers. Receiver operating characteristic curves were used to determine the optimal subcomponent cut-off scores that identified fallers, and the area under the curve (AUC) was used to assess test accuracy. Results: Data from 72 subjects with COPD (mean age, 70.3 ± 7.4y; mean forced expiratory volume in 1 second, 38.9 ± 15.8% predicted) were analyzed. Two BESTest subcomponents, stability limits/verticality (fallers: 75.4%, non-fallers: 83.8%; p=0.002) and postural responses (fallers: 67.5%, non-fallers: 79.7%; p=0.008) distinguished between fallers and non-fallers. Stability limits/verticality had an AUC of 0.70 and optimal cut-off score of 73.8% for identifying fallers; postural responses had an AUC of 0.67 and optimal cut-off score of 69.4%. Conclusion: The stability limits/verticality and postural responses subcomponents of the BESTest distinguished between fallers and non-fallers with COPD. The stability limits/verticality subcomponent can also be used to identify whether an individual with COPD is at risk of falling using a cut-off score of 73.8%. These findings suggest that specific subcomponents of balance could be targeted to optimize fall risk assessment and prevention in COPD.

Aberrant granulosa cell-fate related to inactivated p53/Rb signaling contributes to granulosa cell tumors and to FOXL2 downregulation in the mouse ovary.

Ovarian granulosa cell tumors (GCTs) are indolent tumors of the ovary affecting women at all ages and potentially displaying late recurrence. Even if there is still little information regarding the mechanisms involved in GCT development and progression, FOXL2 would be a major tumor suppressor gene in granulosa cells. We analyzed the mechanisms underlying GCT initiation and progression by using mice with targeted expression of SV40 large T-antigen in granulosa cells (AT mouse), which develop GCTs. Consistent with patients, AT mice with developing GCTs displayed increased levels in circulating anti-Müllerian hormone (AMH), estradiol and androgens, as well as decreased FOXL2 protein abundance. Very few mice developed metastases (1 out of 30). In situ analyses revealed that GCT initiation resulted from both increased granulosa cell survival and proliferation in large antral follicles. Tumorigenesis was associated with the combined inactivation of p53 and Rb pathways, as shown by the impaired expression of respective downstream targets regulating cell apoptosis and proliferation, i.e., Bax, Bak, Gadd45a, Ccna2, Ccne1, E2f1, and Orc1. Importantly, the expression of FOXL2 was still present in newly developed GCTs and its downregulation only started during GCT growth. Collectively, our experiments provide evidence that disrupted p53/Rb signaling can drive tumor initiation and growth. This model challenges the current paradigm that impaired FOXL2 signaling is a major switch of granulosa cell tumorigenesis, albeit possibly contributing to tumor growth.

Glucocorticoids stimulate hypothalamic dynorphin expression accounting for stress-induced impairment of GnRH secretion during preovulatory period.

Stress-induced reproductive dysfunction is frequently associated with increased glucocorticoid (GC) levels responsible for suppressed GnRH/LH secretion and impaired ovulation. Besides the major role of the hypothalamic kisspeptin system, other key regulators may be involved in such regulatory mechanisms. Herein, we identify dynorphin as a novel transcriptional target of GC. We demonstrate that only priming with high estrogen (E2) concentrations prevailing during the late prooestrus phase enables stress-like GC concentrations to specifically stimulate Pdyn (prodynorphin) expression both in vitro (GT1-7 mouse hypothalamic cell line) and ex vivo (ovariectomized E2-supplemented mouse brains). Our results indicate that stress-induced GC levels up-regulate dynorphin expression within a specific kisspeptin neuron-containing hypothalamic region (antero-ventral periventricular nucleus), thus lowering kisspeptin secretion and preventing preovulatory GnRH/LH surge at the end of the prooestrus phase. To further characterize the molecular mechanisms of E2 and GC crosstalk, chromatin immunoprecipitation experiments and luciferase reporter gene assays driven by the proximal promoter of Pdyn show that glucocorticoid receptors bind specific response elements located within the Pdyn promoter, exclusively in presence of E2. Altogether, our work provides novel understanding on how stress affects hypothalamic-pituitary-gonadal axis and underscores the role of dynorphin in mediating GC inhibitory actions on the preovulatory GnRH/LH surge to block ovulation.

A novel non genomic glucocorticoid signaling mediated by a membrane palmitoylated glucocorticoid receptor cross talks with GnRH in gonadotrope cells.

Glucocorticoid hormones (GC) are the main stress mediators associated with reproductive disorders. GC exert their effects through activation of the glucocorticoid receptor (GR) principally acting as a transcription factor. Beside well-established GR-mediated genomic actions, several lines of evidence suggest a role for rapid membrane-initiated GC signaling in gonadotrope cells triggered by a membrane-associated GR. Herein, we demonstrate the existence of a specific membrane-initiated GC signaling in LßT2 gonadotrope cells involving two related phosphoproteins: Ca2+/Calmodulin-dependent protein kinase II (CaMKII) and synapsin-I. Within 5 min, LßT2 cells treated with stress range of 10-7 M Corticosterone or a membrane impermeable-GC, BSA-conjugated corticosterone, exhibited a 2-fold increase in levels of phospho-CaMKII and phospho-synapsin-I. Biochemical approaches revealed that this rapid signaling is promoted by a palmitoylated GR. Importantly, GC significantly alter GnRH-induced CaMKII phosphorylation, consistent with a novel cross-talk between the GnRH receptor and GC. This negative effect of GC on GnRH signaling was further observed on LH release by mouse pituitary explants. Altogether, our work provides new findings in GC field by bringing novel understanding on how GR integrates plasma membrane, allowing GC membrane-initiated signaling that differs in presence of GnRH to disrupt GnRH-dependent signaling and LH secretion.

Neuronal stathmins: a family of phosphoproteins cooperating for neuronal development, plasticity and regeneration.

Nervous system development, plasticity and regeneration require numerous, coordinated and finely tuned subcellular mechanisms. Phosphoproteins of the stathmin family, originally identified as intracellular signal relay proteins, are mostly or exclusively expressed in the nervous system with a high level of expression during brain development. Vertebrate stathmins 1-4 all possess a C-terminal "stathmin-like domain" that binds or releases tubulin in a phosphorylation dependent way, and hence participates in the control of microtubule dynamics, an essential process for neuronal differentiation. Contrary to stathmin 1, stathmins 2-4 possess an N-terminal extension whose reversible palmitoylation specifically targets them to the Golgi and intracellular membranes. Regulation of stathmins 2-4 palmitoylation is therefore an important regulatory mechanism that controls their shuttling to various neuronal compartments where they can then act locally. Expression of stathmins is upregulated during neuronal differentiation and plasticity, and altered in numerous neurodegenerative diseases. Experimental perturbation of stathmins expression in Drosophila or in neurons in culture revealed their importance in neuronal growth and differentiation, each stathmin fulfilling at least partially distinct and likely complementary roles. On the other hand, knock-out of stathmins in mice, with the exception of stathmin 2, resulted in mostly mild or no detected phenotype, revealing likely compensations among stathmins. Altogether, through their combinatorial expression and regulation by phosphorylation and by palmitoylation, and through their interactions with tubulin and other neuronal protein targets, the various stathmins appear as essential regulators of neuronal differentiation at the various stages during development and plasticity of the nervous system.

Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases.

Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.

SCLIP is crucial for the formation and development of the Purkinje cell dendritic arbor.

Cerebellar Purkinje cells elaborate one of the most complex dendritic arbors among neurons to integrate the numerous signals they receive from the cerebellum circuitry. Their dendritic differentiation undergoes successive, tightly regulated phases of development involving both regressive and growth events. Although many players regulating the late phases of Purkinje cell dendritogenesis have been identified, intracellular factors controlling earlier phases of dendritic development remain mostly unknown. In this study, we explored the biological properties and functions of SCLIP, a protein of the stathmin family, in Purkinje cell dendritic differentiation and cerebellum development. Unlike the other stathmins, SCLIP is strongly expressed in Purkinje cells during cerebellar development and accumulates in their dendritic processes at a critical period of their formation and outgrowth. To reveal SCLIP functions, we developed a lentiviral-mediated approach on cerebellar organotypic cultures to inhibit or increase its expression in Purkinje cells in their tissue environment. Depletion of SCLIP promoted retraction of the Purkinje cell primitive process and then prevented the formation of new dendrites at early stages of postnatal development. It also prevented their elongation and branching at later phases of differentiation. Conversely, SCLIP overexpression promoted dendritic branching and development. Together, our results demonstrate for the first time that SCLIP is crucial for both the formation and proper development of Purkinje cell dendritic arbors. SCLIP appears thus as a novel and specific factor that controls the early phases of Purkinje cell dendritic differentiation during cerebellum development.

Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting.

BACKGROUND INFORMATION: Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule-regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3'/RB3'' are localized to the Golgi and vesicle-like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. RESULTS: We show that their conserved N-terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle-like localization. Using palmitoylation-deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co-operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin-like domain) extension. CONCLUSIONS: Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.

Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea.

Scanning DNA sequences for mutations and polymorphisms has become one of the most challenging, often expensive and time-consuming obstacles in many molecular genetic applications, including reverse genetic and clinical diagnostic applications. Enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA at the mismatch sites. These methods are often limited by the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in a pool of DNA and their costs. Here, we present detailed biochemical analysis of five Arabidopsis putative mismatch-specific endonucleases. One of them, ENDO1, is presented as the first endonuclease that recognizes and cleaves all types of mismatches with high efficiency. We report on a very simple protocol for the expression and purification of ENDO1. The ENDO1 system could be exploited in a wide range of mutation diagnostic tools. In particular, we report the use of ENDO1 for discovery of point mutations in the gibberellin 3beta-hydrolase gene of Pisum sativum. Twenty-one independent mutants were isolated, five of these were characterized and two new mutations affecting internodes length were identified. To further evaluate the quality of the mutant population we screened for mutations in four other genes and identified 5-21 new alleles per target. Based on the frequency of the obtained alleles we concluded that the pea population described here would be suitable for use in a large reverse-genetics project.

Two separate motifs cooperate to target stathmin-related proteins to the Golgi complex.

The appropriate targeting of membrane-associated proteins involves a diversity of motifs including post-translational modifications and specific protein sequences. Phosphoproteins of the stathmin family are important regulators of microtubule dynamics, in particular in the developing and mature nervous system. Whereas stathmin is cytosolic, SCG10, SCLIP and the splice variants RB3/RB3'/RB3'' are associated with Golgi and vesicular membranes, through their palmitoylated N-terminal A domains. In order to identify essential motifs involved in this specific targeting, we examined the subcellular distribution of various subdomains derived from domain A of SCG10 fused with GFP. We show that the Golgi localization of SCG10 results from the cooperation of two motifs: a membrane-anchoring palmitoylation motif and a newly identified Golgi-specifying sequence. The latter displayed no targeting activity by itself, but retained a Golgi-specifying activity when associated with another membrane-anchoring palmitoylation motif derived from the protein GAP-43. We further identified critical residues for the specific Golgi targeting of domain A. Altogether, our results give new insight into the regulation of the subcellular localization of stathmin family proteins, an important feature of their physiological functions in differentiating and mature neural cells. More generally we provide new information on essential mechanisms of functional protein subcellular targeting.

T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites.

A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2-3 base pairs long, that preferentially includes a G close to the right end of the T-DNA.