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BackgroundCardiovascular disease (CVD) risk factors are highly prevalent among Hispanic/Latino adults, while the prevalence of MRI infarcts is not well-documented. We, therefore, sought to examine the relationships between CVD risk factors and infarcts with brain structure among Hispanic/Latino individuals.MethodsParticipants included 1,886 Hispanic/Latino adults (50–85 years) who underwent magnetic resonance imaging (MRI) as part of the Study of Latinos—Investigation of Neurocognitive Aging-MRI (SOL-INCA-MRI) study. CVD risk was measured approximately 10.5 years before MRI using the Framingham cardiovascular risk score, a measure of 10-year CVD risk (low (

Introduction: HCHS/SOL is a representative study of Hispanic/Latinos living in the US. SOL-INCA examines cognition amongst those of HCHS/SOL over age 50 and SOL-INCA-MRI obtains quantitative MRI measures on a subgroup of these individuals. Prior research in SOL-INCA found that vascular risk factors summarized by the Framingham Cardiovascular Risk Score (Fram CVD) is associated with Mild Cognitive Impairment (MCI)1. We hypothesize that the extent of white matter hyperintensities (WMH) will partially mediate this impact of Fram CVD on MCI prevalence in this cohort. Methods: SOL-INCA-MRI consists of 2366 individuals of Hispanic/Latino Heritage from 4 centers across the US. Demographics of the cohort are summarized in the Table. High resolution MRI were acquired and WMH burden measured by previously reported methods2. WMH volumes were natural log transformed and corrected for scanner type using NeuroCombat. General linear models were used to test the associated between diagnosis (normal, questionable impairment and MCI) and Fram CVD and WMH adjusting for age, gender, education, heritage, and center. Casual mediation analysis was also performed to assess the extent to which WMH mediated the association between Fram CVD and diagnosis. Results: Subjects were 64.6 + 6.8 years of age at MRI, 68.5% were female, 16% had questionable impairment and 13% had MCI. Mean Fram CVD risk was 11.4 + 0.9%. Mean log WMH was -0.13 +1.55. Diagnosis was significantly associated with Fram CVD (beta= 780, p

Introduction: HCHS/SOL is a representative study of Hispanic/Latinos living in the US. HCHS/SOL participants have a high prevalence of diabetes (1) and diabetic associated cognitive decline (2). Previous studies of predominantly non-Hispanic White populations indicate that silent brain infarcts are associated with incident dementia (3). Given that diabetes is prevalent in HCHS/SOL and associated with cognitive decline, we examined whether diabetes may also lead to infarction on MRI. Methods: SOL-INCA-MRI consists of 2668 individuals of Hispanic/Latino Heritage from 4 centers across the US. Demographics of the cohort are summarized in the Table. The presence or absence of infarction on MRI was determined from review of high resolution T1, T2 and FLAIR imaging as well as gradient echo imaging by a neuroradiologist and a stroke neurologist with a high level of agreement (ICC >0.90). Desperate readings were adjudicated by a neurologist with experience in vascular brain injury. Diabetes based on American Diabetes Association definition of normal, prediabetic and diabetic. This definition used measures serum glucose levels adjusted for fasting time and, if available, post-OGTT glucose levels, A1C percentages, and scanned/transcribed anti- diabetic medication use. General linear model was used to test the associated between the presence or absence of MRI and diabetes adjusting for age, gender, heritage, and center. Results: Subjects were 62.3 + 9.2 years of age at MRI range [35-86], 67% were female and the prevalence of MRI infarction was 7% (Table). Pre-diabetes was present in 45% and diabetes in 22%. Stroke prevalence increased with age ranging from

Cortex morphology varies with age, cognitive function, and in neurological and psychiatric diseases. Here the authors report 160 genome-wide significant associations with thickness, surface area and volume of the total cortex and 34 cortical regions from a GWAS meta-analysis in 22,824 adults.

Background: Cervical dystonia is the most common form of focal dystonia. The frequency and pattern of degenerative changes of the cervical spine in patients with cervical dystonia and their relation to clinical symptoms remain unclear as no direct comparison to healthy controls has been performed yet. Here, we used magnetic resonance imaging (MRI) to investigate (1) whether structural abnormalities of the cervical spine are more common in patients with cervical dystonia compared to age-matched healthy controls, (2) if there are clinical predictors for abnormalities on MRI, and (3) to calculate the inter-rater reliability of the respective radiological scales.Methods: Twenty-five consecutive patients with cervical dystonia and 20 age-matched healthy controls were included in the study. MRI scans of the cervical spine were analyzed separately by three experienced raters blinded to clinical information, applying different MRI rating scales. Structural abnormalities were compared between groups for upper, middle, and lower cervical spine segments. The associations between scores differentiating both groups and clinical parameters were assessed in dystonia patients. Additionally, inter-rater reliability of the MRI scales was calculated.Results: Comparing structural abnormalities, we found minor differences in the middle cervical spine, indicated by a higher MRI total score in patients but no significant correlation between clinical parameters and MRI changes. Inter-rater reliability was satisfying for most of the MRI rating scales.Conclusion: Our results do not provide evidence for a role of MRI of the cervical spine in the routine work-up of patients with cervical dystonia in the absence of specific clinical signs or symptoms.

MRI studies have consistently identified atrophy patterns in Alzheimer’s disease (AD) through a whole-brain voxel-based analysis, but efforts to investigate morphometric profiles using anatomically standardized and automated whole-brain ROI analyses, performed at the individual subject space, are still lacking. In this study we aimed (i) to utilize atlas-derived measurements of cortical thickness and subcortical volumes, including of the hippocampal subfields, to identify atrophy patterns in early-stage AD, and (ii) to compare cognitive profiles at baseline and during a one-year follow-up of those previously identified morphometric AD subtypes to predict disease progression. Through a prospectively recruited multi-center study, conducted at four Austrian sites, 120 patients were included with probable AD, a disease onset beyond 60 years and a clinical dementia rating of ≤1. Morphometric measures of T1-weighted images were obtained using FreeSurfer. A principal component and subsequent cluster analysis identified four morphometric subtypes, including (i) hippocampal predominant (30.8%), (ii) hippocampal-temporo-parietal (29.2%), (iii) parieto-temporal (hippocampal sparing, 20.8%) and (iv) hippocampal-temporal (19.2%) atrophy patterns that were associated with phenotypes differing predominately in the presentation and progression of verbal memory and visuospatial impairments. These morphologically distinct subtypes are based on standardized brain regions, which are anatomically defined and freely accessible so as to validate its diagnostic accuracy and enhance the prediction of disease progression.

Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively.

Magnetic resonance imaging (MRI) is sensitive to structural and functional changes in the brain caused by Alzheimer's disease (AD), and can therefore be used to help in diagnosing the disease. Improving classification of AD patients based on MRI scans might help to identify AD earlier in the disease's progress, which may be key in developing treatments for AD. In this study we used an elastic net classifier based on several measures derived from the MRI scans of mild to moderate AD patients (N=77) from the prospective registry on dementia study and controls (N=173) from the Austrian Stroke Prevention Family Study. We based our classification on measures from anatomical MRI, diffusion weighted MRI and resting state functional MRI. Our unimodal classification performance ranged from an area under the curve (AUC) of 0.760 (full correlations between functional networks) to 0.909 (grey matter density). When combining measures from multiple modalities in a stepwise manner, the classification performance improved to an AUC of 0.952. This optimal combination consisted of grey matter density, white matter density, fractional anisotropy, mean diffusivity, and sparse partial correlations between functional networks. Classification performance for mild AD as well as moderate AD also improved when using this multimodal combination. We conclude that different MRI modalities provide complementary information for classifying AD. Moreover, combining multiple modalities can substantially improve classification performance over unimodal classification.

BACKGROUND:Quantitative susceptibility mapping (QSM) and R2* relaxation rate mapping have demonstrated increased iron deposition in the substantia nigra of patients with idiopathic Parkinson's disease (PD). However, the findings in other subcortical deep gray matter nuclei are converse and the sensitivity of QSM and R2* for morphological changes and their relation to clinical measures of disease severity has so far been investigated only sparsely. METHODS:The local ethics committee approved this study and all subjects gave written informed consent. 66 patients with idiopathic Parkinson's disease and 58 control subjects underwent quantitative MRI at 3T. Susceptibility and R2* maps were reconstructed from a spoiled multi-echo 3D gradient echo sequence. Mean susceptibilities and R2* rates were measured in subcortical deep gray matter nuclei and compared between patients with PD and controls as well as related to clinical variables. RESULTS:Compared to control subjects, patients with PD had increased R2* values in the substantia nigra. QSM also showed higher susceptibilities in patients with PD in substantia nigra, in the nucleus ruber, thalamus, and globus pallidus. Magnetic susceptibility of several of these structures was correlated with the levodopa-equivalent daily dose (LEDD) and clinical markers of motor and non-motor disease severity (total MDS-UPDRS, MDS-UPDRS-I and II). Disease severity as assessed by the Hoehn & Yahr scale was correlated with magnetic susceptibility in the substantia nigra. CONCLUSION:The established finding of higher R2* rates in the substantia nigra was extended by QSM showing superior sensitivity for PD-related tissue changes in nigrostriatal dopaminergic pathways. QSM additionally reflected the levodopa-dosage and disease severity. These results suggest a more widespread pathologic involvement and QSM as a novel means for its investigation, more sensitive than current MRI techniques.

Cerebral white matter lesions (WML) have been found in normal aging, vascular disease and several neuropsychiatric conditions. Correlations of WML with clinical parameters in BD have been described, but not with the number of affective episodes, illness duration, age of onset and Body Mass Index in a well characterized group of euthymic bipolar adults. Herein, we aimed to evaluate the associations between bipolar course of illness parameters and WML measured with volumetric analysis.In a cross-sectional study 100 euthymic individuals with BD as well as 54 healthy controls (HC) were enrolled to undergo brain magnetic resonance imaging using 3T including a FLAIR sequence for volumetric assessment of WML-load using FSL-software. Additionally, clinical characteristics and psychometric measures including Structured Clinical Interview according to DSM-IV, Hamilton-Depression, Young Mania Rating Scale and Beck's Depression Inventory were evaluated.Individuals with BD had significantly more (F = 3.968, p < .05) WML (Mdn = 3710 mm3; IQR = 2961 mm3) than HC (Mdn = 2185 mm3; IQR = 1665 mm3). BD men (Mdn = 4095 mm3; IQR = 3295 mm3) and BD women (Mdn = 3032 mm3; IQR = 2816 mm3) did not significantly differ as to the WML-load or the number and type of risk factors for WML. However, in men only, the number of manic/hypomanic episodes (r = 0.72; p < .001) as well as depressive episodes (r = 0.51; p < .001) correlated positively with WML-load.WML-load strongly correlated with the number of manic episodes in male BD patients, suggesting that men might be more vulnerable to mania in the context of cerebral white matter changes.

Analysis of nonlinear quantitative EEG (qEEG) markers describing complexity of signal in relation to severity of Alzheimer’s disease (AD) was the focal point of this study. In this study, 79 patients diagnosed with probable AD were recruited from the multi-centric Prospective Dementia Database Austria (PRODEM). EEG recordings were done with the subjects seated in an upright position in a resting state with their eyes closed. Models of linear regressions explaining disease severity, expressed in Mini Mental State Examination (MMSE) scores, were analyzed by the nonlinear qEEG markers of auto mutual information (AMI), Shannon entropy (ShE), Tsallis entropy (TsE), multiscale entropy (MsE), or spectral entropy (SpE), with age, duration of illness, and years of education as co-predictors. Linear regression models with AMI were significant for all electrode sites and clusters, where R 2 is 0.46 at the electrode site C3, 0.43 at Cz, F3, and central region, and 0.42 at the left region. MsE also had significant models at C3 with R 2 > 0.40 at scales τ = 5 and τ = 6 . ShE and TsE also have significant models at T7 and F7 with R 2 > 0.30 . Reductions in complexity, calculated by AMI, SpE, and MsE, were observed as the MMSE score decreased.

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.

Septum formation in Neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, BUD-4, and CHS-1. In chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (SAT) assembly, 2) contractile actomyosin ring (CAR) formation, 3) CAR constriction together with plasma membrane ingrowth and cell wall construction. Septation began with the assembly of a conspicuous tangle of cortical actin cables (SAT) in the septation site >5 min before plasma membrane ingrowth. Tropomyosin and myosin were detected as components of the SAT from the outset. The SAT gradually condensed to form a proto-CAR that preceded CAR formation. During septum development, the contractile actomyosin ring remained associated with the advancing edge of the septum. Formin and BUD-4 were recruited during the transition from SAT to CAR and CHS-1 appeared two min before CAR constriction. Actin patches containing fimbrin were observed surrounding the ingrowing septum, an indication of endocytic activity. Although the trigger of SAT assembly remains unclear, the regularity of septation both in space and time gives us reason to believe that the initiation of the septation process is integrated with the mechanisms that control both the cell cycle and the overall growth of hyphae, despite the asynchronous nature of mitosis in N. crassa.

Intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. Cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. Approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the model filamentous fungus Neurospora crassa. Genetic, biochemical, and microscopic techniques were used to characterize the functions of seven previously poorly characterized cell fusion proteins. HAM-6, HAM-7 and HAM-8 share functional characteristics and are proposed to function in the same signaling network. Our data suggest that these proteins may form a sensor complex at the cell wall/plasma membrane for the MAK-1 cell wall integrity mitogen-activated protein kinase (MAPK) pathway. We also demonstrate that HAM-9, HAM-10, AMPH-1 and WHI-2 have more general functions and are required for normal growth and development. The activation status of the MAK-1 and MAK-2 MAPK pathways are altered in mutants lacking these proteins. We propose that these proteins may function to coordinate the activities of the two MAPK modules with other signaling pathways during cell fusion.

Proper cell division is essential for growth and development of uni- and multicellular organisms. The fungal septation initiation network (SIN) functions as kinase cascade that connects cell cycle progression with the initiation of cytokinesis. Miss-regulation of the homologous Hippo pathway in animals results in excessive cell proliferation and formation of tumors, underscoring the conservation of both pathways. How SIN proteins interact and transmit signals through the cascade is only beginning to be understood. Moreover, our understanding of septum formation and its regulation in filamentous fungi, which represent the vast majority of the fungal kingdom, is highly fragmentary. We determined that a tripartite kinase cascade, consisting of CDC-7, SID-1 and DBF-2, together with their regulatory subunits CDC-14 and MOB-1, is important for septum formation in the model mold Neurospora crassa. DBF-2 activity and septum formation requires auto-phosphorylation at Ser499 within the activation segment and phosphorylation of Thr671 in the hydrophobic motif by SID-1. Moreover, SID-1-stimulated DBF-2 activity is further enhanced by CDC-7, supporting a stepwise activation mechanism of the tripartite SIN kinase cascade in fungi. However, in contrast to the situation described for unicellular yeasts, the localization of the entire SIN cascade to spindle pole bodies is constitutive and cell cycle independent. Moreover, all SIN proteins except CDC-7 form cortical rings prior to septum initiation and localize to constricting septa. Thus, SIN localization and activity regulation significantly differs in unicellular versus syncytial ascomycete fungi.

Cell communication is essential for eukaryotic development, but our knowledge of molecules and mechanisms required for intercellular communication is fragmentary. In particular, the connection between signal sensing and regulation of cell polarity is poorly understood. In the filamentous ascomycete Neurospora crassa, germinating spores mutually attract each other and subsequently fuse. During these tropic interactions, the two communicating cells rapidly alternate between two different physiological states, probably associated with signal delivery and response. The MAK2 MAP kinase cascade mediates cell-cell signaling. Here, we show that the conserved scaffolding protein HYM1/MO25 controls the cell shape-regulating NDR kinase module as well as the signal-receiving MAP kinase cascade. HYM1 functions as an integral part of the COT1 NDR kinase complex to regulate the interaction with its upstream kinase POD6 and thereby COT1 activity. In addition, HYM1 interacts with NRC1, MEK2, and MAK2, the three kinases of the MAK2 MAP kinase cascade, and co-localizes with MAK2 at the apex of growing cells. During cell fusion, the three kinases of the MAP kinase module as well as HYM1 are recruited to the point of cell-cell contact. hym-1 mutants phenocopy all defects observed for MAK2 pathway mutants by abolishing MAK2 activity. An NRC1-MEK2 fusion protein reconstitutes MAK2 signaling in hym-1, while constitutive activation of NRC1 and MEK2 does not. These data identify HYM1 as a novel regulator of the NRC1-MEK2-MAK2 pathway, which may coordinate NDR and MAP kinase signaling during cell polarity and intercellular communication.

OBJECTIVE: To assess the influence of cognitive, functional and behavioral factors, co-morbidities as well as caregiver characteristics on driving cessation in dementia patients. METHODS: The study cohort consists of those 240 dementia cases of the ongoing prospective registry on dementia in Austria (PRODEM) who were former or current car-drivers (mean age 74.2 (±8.8) years, 39.6% females, 80.8% Alzheimer's disease). Reasons for driving cessation were assessed with the patients' caregivers. Standardized questionnaires were used to evaluate patient- and caregiver characteristics. Cognitive functioning was determined by Mini-Mental State Examination (MMSE), the CERAD neuropsychological test battery and Clinical Dementia Rating (CDR), activities of daily living (ADL) by the Disability Assessment for Dementia, behavior by the Neuropsychiatric Inventory (NPI) and caregiver burden by the Zarit burden scale. RESULTS: Among subjects who had ceased driving, 136 (93.8%) did so because of "Unacceptable risk" according to caregiver's judgment. Car accidents and revocation of the driving license were responsible in 8 (5.5%) and 1(0.7%) participant, respectively. Female gender (OR 5.057; 95%CI 1.803-14.180; p = 0.002), constructional abilities (OR 0.611; 95%CI 0.445-0.839; p = 0.002) and impairment in Activities of Daily Living (OR 0.941; 95%CI 0.911-0.973; p

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P(2) that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.

Rho-type GTPases are key regulators that control eukaryotic cell polarity, but their role in fungal morphogenesis is only beginning to emerge. In this study, we investigate the role of the CDC-42 - RAC - CDC-24 module in Neurospora crassa. rac and cdc-42 deletion mutants are viable, but generate highly compact colonies with severe morphological defects. Double mutants carrying conditional and loss of function alleles of rac and cdc-42 are lethal, indicating that both GTPases share at least one common essential function. The defects of the GTPase mutants are phenocopied by deletion and conditional alleles of the guanine exchange factor (GEF) cdc-24, and in vitro GDP-GTP exchange assays identify CDC-24 as specific GEF for both CDC-42 and RAC. In vivo confocal microscopy shows that this module is organized as membrane-associated cap that covers the hyphal apex. However, the specific localization patterns of the three proteins are distinct, indicating different functions of RAC and CDC-42 within the hyphal tip. CDC-42 localized as confined apical membrane-associated crescent, while RAC labeled a membrane-associated ring excluding the region labeled by CDC42. The GEF CDC-24 occupied a strategic position, localizing as broad apical membrane-associated crescent and in the apical cytosol excluding the Spitzenkörper. RAC and CDC-42 also display distinct localization patterns during branch initiation and germ tube formation, with CDC-42 accumulating at the plasma membrane before RAC. Together with the distinct cellular defects of rac and cdc-42 mutants, these localizations suggest that CDC-42 is more important for polarity establishment, while the primary function of RAC may be maintaining polarity. In summary, this study identifies CDC-24 as essential regulator for RAC and CDC-42 that have common and distinct functions during polarity establishment and maintenance of cell polarity in N. crassa.

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.