Mechanistic Basis of Branch-Site Selection in Filamentous Bacteria

Many filamentous organisms, such as fungi, grow by tip-extension and by forming new branches behind the tips. A similar growth mode occurs in filamentous bacteria, including the genus Streptomyces, although here our mechanistic understanding has been very limited. The Streptomyces protein DivIVA is a critical determinant of hyphal growth and localizes in foci at hyphal tips and sites of future branch development. However, how such foci form was previously unknown. Here, we show experimentally that DivIVA focus-formation involves a novel mechanism in which new DivIVA foci break off from existing tip-foci, bypassing the need for initial nucleation or de novo branch-site selection. We develop a mathematical model for DivIVA-dependent growth and branching, involving DivIVA focus-formation by tip-focus splitting, focus growth, and the initiation of new branches at a critical focus size. We quantitatively fit our model to the experimentally-measured tip-to-branch and branch-to-branch length distributions. The model predicts a particular bimodal tip-to-branch distribution results from tip-focus splitting, a prediction we confirm experimentally. Our work provides mechanistic understanding of a novel mode of hyphal growth regulation that may be widely employed

Regulation of apical growth and hyphal branching in Streptomyces

The filamentous bacteria Streptomyces grow by tip extension and through the initiation of new branches, and this apical growth is directed by a polarisome-like complex involving the essential polarity protein DivIVA. New branch sites must be marked de novo and, until recently, there was no understanding of how these new sites are selected. Equally, hyphal branching patterns are affected by environmental conditions, but there was no insight into how polar growth and hyphal branching might be regulated in response to external or internal cues. This review focuses on recent discoveries that reveal the principal mechanism of branch site selection in Streptomyces, and the first mechanism to be identified that regulates polarisome behaviour to modulate polar growth and hyphal branching

Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of Streptomyces coelicolor by Targeted LC-MS/MS

The filamentous bacterium Streptomyces coelicolor modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC-MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC-MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD00009S

Main parameters and their values.

<p>Main parameters and their values.</p

Evidence of tip-focus splitting, growth of foci and emergence of branches, in fluorescence-imaged <i>Streptomyces coelicolor</i> expressing <i>divIVA-egfp</i>.

<p>The tip always contains a large DivIVA focus and established tips extend at an approximately constant speed. At about 12 minutes, the DivIVA tip-focus undergoes splitting, leaving behind a new focus (arrow). As the tip continues to extend, the new focus remains in place on the membrane and grows in intensity. After about 42 minutes a new branch is formed at the position of the new focus, with the new focus now sitting at the tip of the new branch. Both the new branch and the original branch now continue to extend in length. Time in hours∶minutes. Scale bar: .</p

Determination of Phosphorylation Sites in the DivIVA Cytoskeletal Protein of <i>Streptomyces coelicolor</i> by Targeted LC–MS/MS

The filamentous bacterium <i>Streptomyces coelicolor</i> modulates polar growth and branching by phosphorylating the cytoskeletal protein DivIVA. Previous MALDI-TOF analysis of DivIVA showed that a large 7.2 kDa tryptic peptide was multiply phosphorylated. To aid localization of the phosphorylation sites, we introduced additional tryptic cleavage sites into DivIVA, and the resulting phosphopeptides were analyzed by LC–MS/MS. Phosphopeptide isomers could be separated chromatographically, but because of overlapping elution and spectrum quality, site assignment by standard software tools was ambiguous. Because fragment ions carrying the phosphate group are essential for confident localization, large numbers of spectra were collected using targeted LC–MS/MS, and a special script was developed for plotting the elution of site-determining fragments from those spectra under the XIC of the parent ions. Where multiple phosphopeptide isomers were present, the elution of the site-determining y-ions perfectly coincided with the elution of the corresponding phosphopeptide isomer. This method represents a useful tool for user inspection of spectra derived from phosphopeptide isomers and significantly increases confidence when defining phosphorylation sites. In this way, we show that DivIVA is phosphorylated in vivo on five sites in the C-terminal part of the protein (T304, S309, S338, S344, and S355). The data have been deposited to the ProteomeXchange Consortium with identifier PXD000095

The course of cancer-related insomnia: don't expect it to disappear after cancer treatment

Objective: The study aimed to examine the 12-month course of cancer-related insomnia (CRI) and to identify possible predictors for the prevalence and persistence of CRI. Methods: This longitudinal multicenter study included N = 405 patients with cancer (56% females, mean age: 58.6 years). CRI was measured by the Insomnia Severity Index (ISI). Socio-demographic and clinical data, as well as psychological parameters (Distress Thermometer, PHQ-9, GAD-7, and EORTC-Fatigue), were assessed at baseline (T1) and 12 months later (T2). Results: In our sample, a high prevalence of relevant insomnia symptoms (49.4%, ISI > 7) was found, while a clinical insomnia diagnosis was verified in 12.8% (ISI > 14). When insomnia was present at T1, this problem was persistent after one year in 64%. At T2, however, significantly more women suffered from insomnia symptoms (53.3% women vs. 39.3% men; p = 0.003). Insomnia was associated with many clinical and psychological parameters, especially with fatigue (r = 0.5). Multiple regression analysis revealed that, in women, only insomnia at T1 was a significant predictor for insomnia at T2 (R-2 = 0.40; F(5) = 12.5; p < 0.001), whereas in men insomnia, depressive symptoms and the use of psychotropic drugs at T1 predicted the extent of insomnia at T2 (R-2 = 0.28; F(7) = 9.5; p < 0.001). In all participants, levels of distress, depression, and anxiety decreased from T1 to T2 (p's < 0.016). Conclusion: Insomnia is a common disorder in cancer patients. Although medical and psychological parameters improved during the 12-month course of cancer treatment, our results show that insomnia is highly persistent, especially in women. This indicates that adequate support for those affected is needed. (C) 2019 Elsevier B.V. All rights reserved

The Ser/Thr protein kinase AfsK regulates polar growth and hyphal branching in the filamentous bacteria Streptomyces.

In cells that exhibit apical growth, mechanisms that regulate cell polarity are crucial for determination of cellular shape and for the adaptation of growth to intrinsic and extrinsic cues. Broadly conserved pathways control cell polarity in eukaryotes, but less is known about polarly growing prokaryotes. An evolutionarily ancient form of apical growth is found in the filamentous bacteria Streptomyces, and is directed by a polarisome-like complex involving the essential protein DivIVA. We report here that this bacterial polarization machinery is regulated by a eukaryotic-type Ser/Thr protein kinase, AfsK, which localizes to hyphal tips and phosphorylates DivIVA. During normal growth, AfsK regulates hyphal branching by modulating branch-site selection and some aspect of the underlying polarisome-splitting mechanism that controls branching of Streptomyces hyphae. Further, AfsK is activated by signals generated by the arrest of cell wall synthesis and directly communicates this to the polarisome by hyperphosphorylating DivIVA. Induction of high levels of DivIVA phosphorylation by using a constitutively active mutant AfsK causes disassembly of apical polarisomes, followed by establishment of multiple hyphal branches elsewhere in the cell, revealing a profound impact of this kinase on growth polarity. The function of AfsK is reminiscent of the phoshorylation of polarity proteins and polarisome components by Ser/Thr protein kinases in eukaryotes

Return to work after cancer: Improved mental health in working cancer survivors

Objective Aim of the study was to compare working and non-working patients over a period of 12 months regarding socio-demographic, cancer-specific and mental health parameters. Methods This study was conducted as part of a Germany-wide longitudinal survey among 1398 patients in 13 national Comprehensive Cancer Centers. The sample used for analysis consisted of n = 430 cancer patients younger than 65 years (age M = 52.4 years, SD = 8.1; 67.0% females). Socio-demographic, cancer-specific and mental health parameters (Depression: Patient Health Questionnaire, Anxiety: Generalized Anxiety Disorder Scale, Distress: Distress Thermometer) were assessed at baseline during hospitalization and at 12 months follow-up. Results 73.7% of all patients (n = 317) have returned to work after one year. While working and non-working patients did not differ in socio-demographic parameters, there were significant differences in the presence of metastases, tumor and treatment status. Mixed analysis of variances revealed significant interactions between working status and time for depression (p = 0.009), anxiety (p = 0.003) and distress (p = 0.007). Non-working patients reported higher levels of depression, anxiety and distress than working patients over time. A logistic regression showed significant associations between lower depression (p = 0.019), lower distress (p = 0.033) and the absence of a tumor (p = 0.015) with working status. Conclusions The majority of cancer survivors returned to work. Non-working patients had higher levels of depression, anxiety and distress than working patients. After controlling for cancer-specific factors, mental health parameters were still independently associated with working status. Return to work can thus be associated with an improved mental health in cancer survivors. In order to establish causality, further research is necessary