CED-6/GULP and Components of the Clathrin-mediated Endocytosis Machinery Act Redundantly to Correctly Display CED-1 on the Cell Membrane in C. elegans.

CED-1 is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

Covert Consciousness in Acute Brain Injury Revealed by Automated Pupillometry and Cognitive Paradigms.

BACKGROUND: Identifying covert consciousness in intensive care unit (ICU) patients with coma and other disorders of consciousness (DoC) is crucial for treatment decisions, but sensitive low-cost bedside markers are missing. We investigated whether automated pupillometry combined with passive and active cognitive paradigms can detect residual consciousness in ICU patients with DoC. METHODS: We prospectively enrolled clinically low-response or unresponsive patients with traumatic or nontraumatic DoC from ICUs of a tertiary referral center. Age-matched and sex-matched healthy volunteers served as controls. Patients were categorized into clinically unresponsive (coma or unresponsive wakefulness syndrome) or clinically low-responsive (minimally conscious state or better). Using automated pupillometry, we recorded pupillary dilation to passive (visual and auditory stimuli) and active (mental arithmetic) cognitive paradigms, with task-specific success criteria (e.g., ≥ 3 of 5 pupillary dilations on five consecutive mental arithmetic tasks). RESULTS: We obtained 699 pupillometry recordings at 178 time points from 91 ICU patients with brain injury (mean age 60 ± 13.8 years, 31% women, and 49.5% nontraumatic brain injuries). Recordings were also obtained from 26 matched controls (59 ± 14.8 years, 38% women). Passive paradigms yielded limited distinctions between patients and controls. However, active paradigms enabled discrimination between different states of consciousness. With mental arithmetic of moderate complexity, ≥ 3 pupillary dilations were seen in 17.8% of clinically unresponsive patients and 50.0% of clinically low-responsive patients (odds ratio 4.56, 95% confidence interval 2.09-10.10; p < 0.001). In comparison, 76.9% healthy controls responded with ≥ 3 pupillary dilations (p = 0.028). Results remained consistent across sensitivity analyses using different thresholds for success. Spearman's rank analysis underscored the robust association between pupillary dilations during mental arithmetic and consciousness levels (rho = 1, p = 0.017). Notably, one behaviorally unresponsive patient demonstrated persistent command-following behavior 2 weeks before overt signs of awareness, suggesting prolonged cognitive motor dissociation. CONCLUSIONS: Automated pupillometry combined with mental arithmetic can identify cognitive efforts, and hence covert consciousness, in ICU patients with acute DoC.

Multimodal Prediction of 3- and 12-Month Outcomes in ICU Patients with Acute Disorders of Consciousness.

BACKGROUND: In intensive care unit (ICU) patients with coma and other disorders of consciousness (DoC), outcome prediction is key to decision-making regarding prognostication, neurorehabilitation, and management of family expectations. Current prediction algorithms are largely based on chronic DoC, whereas multimodal data from acute DoC are scarce. Therefore, the Consciousness in Neurocritical Care Cohort Study Using Electroencephalography and Functional Magnetic Resonance Imaging (i.e. CONNECT-ME; ClinicalTrials.gov identifier: NCT02644265) investigates ICU patients with acute DoC due to traumatic and nontraumatic brain injuries, using electroencephalography (EEG) (resting-state and passive paradigms), functional magnetic resonance imaging (fMRI) (resting-state) and systematic clinical examinations. METHODS: We previously presented results for a subset of patients (n = 87) concerning prediction of consciousness levels in the ICU. Now we report 3- and 12-month outcomes in an extended cohort (n = 123). Favorable outcome was defined as a modified Rankin Scale score ≤ 3, a cerebral performance category score ≤ 2, and a Glasgow Outcome Scale Extended score ≥ 4. EEG features included visual grading, automated spectral categorization, and support vector machine consciousness classifier. fMRI features included functional connectivity measures from six resting-state networks. Random forest and support vector machine were applied to EEG and fMRI features to predict outcomes. Here, random forest results are presented as areas under the curve (AUC) of receiver operating characteristic curves or accuracy. Cox proportional regression with in-hospital death as a competing risk was used to assess independent clinical predictors of time to favorable outcome. RESULTS: Between April 2016 and July 2021, we enrolled 123 patients (mean age 51 years, 42% women). Of 82 (66%) ICU survivors, 3- and 12-month outcomes were available for 79 (96%) and 77 (94%), respectively. EEG features predicted both 3-month (AUC 0.79 [95% confidence interval (CI) 0.77-0.82]) and 12-month (AUC 0.74 [95% CI 0.71-0.77]) outcomes. fMRI features appeared to predict 3-month outcome (accuracy 0.69-0.78) both alone and when combined with some EEG features (accuracies 0.73-0.84) but not 12-month outcome (larger sample sizes needed). Independent clinical predictors of time to favorable outcome were younger age (hazard ratio [HR] 1.04 [95% CI 1.02-1.06]), traumatic brain injury (HR 1.94 [95% CI 1.04-3.61]), command-following abilities at admission (HR 2.70 [95% CI 1.40-5.23]), initial brain imaging without severe pathological findings (HR 2.42 [95% CI 1.12-5.22]), improving consciousness in the ICU (HR 5.76 [95% CI 2.41-15.51]), and favorable visual-graded EEG (HR 2.47 [95% CI 1.46-4.19]). CONCLUSIONS: Our results indicate that EEG and fMRI features and readily available clinical data predict short-term outcome of patients with acute DoC and that EEG also predicts 12-month outcome after ICU discharge.

Human calmodulin mutations cause arrhythmia and affect neuronal function in C. elegans.

In humans, mutations in calmodulin cause cardiac arrhythmia. These mutations disrupt the ability of calmodulin to sense calcium concentrations and correctly regulate two central calcium channels, together obstructing heart rhythm. This correlation is well established, but also surprising since calmodulin is expressed in all tissues and interacts with hundreds of proteins. Until now, most studies have focused on cardiac cell function and regulation of specific cardiac targets, and thus, potential other effects of these mutations have largely been unexplored. Here, we introduce the nematode Caenorhabditis elegans as an in vivo model to study effects of three human calmodulin mutations with different impairment on calcium binding. We find that arrhythmic effects of the calmodulin mutations N54I and D96V can be recapitulated in disruption of two rhythmic behaviors, pharynx pumping and defecation motor program. Interestingly, we also find that these mutations affect neuronal function, but in different ways. Whereas D96V sensitizes signaling at the neuromuscular junction, N54I has a protective effect. The mutation N98S did not affect rhythmic behavior, but impaired chemosensing. Therefore, pathogenic calmodulin mutations act through different mechanisms in rhythmic behavior and neuronal function in C. elegans, emphasizing the strength of using live multicellular models. Finally, our results support the hypothesis that human calmodulin mutations could also contribute to neurological diseases.

Multimodal prediction of residual consciousness in the intensive care unit: the CONNECT-ME study.

Functional MRI (fMRI) and EEG may reveal residual consciousness in patients with disorders of consciousness (DoC), as reflected by a rapidly expanding literature on chronic DoC. However, acute DoC is rarely investigated, although identifying residual consciousness is key to clinical decision-making in the intensive care unit (ICU). Therefore, the objective of the prospective, observational, tertiary centre cohort, diagnostic phase IIb study 'Consciousness in neurocritical care cohort study using EEG and fMRI' (CONNECT-ME, NCT02644265) was to assess the accuracy of fMRI and EEG to identify residual consciousness in acute DoC in the ICU. Between April 2016 and November 2020, 87 acute DoC patients with traumatic or non-traumatic brain injury were examined with repeated clinical assessments, fMRI and EEG. Resting-state EEG and EEG with external stimulations were evaluated by visual analysis, spectral band analysis and a Support Vector Machine (SVM) consciousness classifier. In addition, within- and between-network resting-state connectivity for canonical resting-state fMRI networks was assessed. Next, we used EEG and fMRI data at study enrolment in two different machine-learning algorithms (Random Forest and SVM with a linear kernel) to distinguish patients in a minimally conscious state or better (≥MCS) from those in coma or unresponsive wakefulness state (≤UWS) at time of study enrolment and at ICU discharge (or before death). Prediction performances were assessed with area under the curve (AUC). Of 87 DoC patients (mean age, 50.0 ± 18 years, 43% female), 51 (59%) were ≤UWS and 36 (41%) were ≥ MCS at study enrolment. Thirty-one (36%) patients died in the ICU, including 28 who had life-sustaining therapy withdrawn. EEG and fMRI predicted consciousness levels at study enrolment and ICU discharge, with maximum AUCs of 0.79 (95% CI 0.77-0.80) and 0.71 (95% CI 0.77-0.80), respectively. Models based on combined EEG and fMRI features predicted consciousness levels at study enrolment and ICU discharge with maximum AUCs of 0.78 (95% CI 0.71-0.86) and 0.83 (95% CI 0.75-0.89), respectively, with improved positive predictive value and sensitivity. Overall, both machine-learning algorithms (SVM and Random Forest) performed equally well. In conclusion, we suggest that acute DoC prediction models in the ICU be based on a combination of fMRI and EEG features, regardless of the machine-learning algorithm used.

Movement Patterns of Roaming Companion Cats in Denmark-A Study Based on GPS Tracking.

We studied the roaming patterns of companion cats in Denmark. The movements of 97 cats with outdoor access were traced for about seven days using GPS tracking. Data on the cats were gathered from their owners. The median time cats spent away from their homes was 5 h per day (IQR: 2.5 to 8.8 h), median daily distance moved was 2.4 km (IQR: 1.3 to 3.7 km), and median for 95% BBKDE home range was 5 ha (IQR: 2.9 to 8.5 ha). Cats above seven years of age spent less time away from home, were less active and had a smaller home range than younger cats. Cats with access to nature areas spent more time away from home, were more active and had larger home ranges. Intact male cats spent more time away from home than neutered cats and had larger home ranges as well. Finally, rainfall had an impact on the distance moved by cats: on days without rainfall the cats moved 3.6 km on average (95% CI: 2.8; 4.5 km); and on days with heavy rainfall the cats moved 2.4 km on average (95% CI: 1.6; 3.5 km).

Estimating the Population of Unowned Free-Ranging Domestic Cats in Denmark Using a Combination of Questionnaires and GPS Tracking.

The present study aimed (1) to estimate the size of the population of unowned free-ranging domestic cats in Denmark using a questionnaire survey combined with a GPS-tracking survey, and (2) to estimate the distribution of the population across different habitats. The questionnaires were circulated in 94 randomly selected parishes ranging across seven kinds of habitat. Using responses from five of the habitats, we estimated the population of unowned free-ranging cats nationally. In the other two habitats, questionnaire data were collected in a simpler way. The territory of 59 owned cats was estimated with GPS tracking to assess home ranges. Home range area was calculated using 95% Brownian bridge kernel density estimation (0.033-0.077 ± 0.011-0.023 km2, median ± SE). We estimated a population of unowned free-ranging cats in Denmark of 89,000 ± 11,000 (SE), with a mean density of 2 ± 0.3 (SE) cats per km2, living primarily in rural habitats. Approximately one-third of the cats were estimated to be socialised and two-thirds unsocialised. Our method may be suitable for use in other temperate areas facing problems with unowned free-ranging cats.

Use of non-insulin antidiabetic drugs in children and young adults - A Scandinavian drug utilization study from 2010-2019.

Knowledge on utilization patterns of non-insulin antidiabetic drugs in childhood and youth is limited. Therefore, we conducted a population-based drug utilization study using publicly available aggregate data on use of non-insulin antidiabetics from 2010 to 2019 in Scandinavia (Denmark, Norway and Sweden) in individuals aged up to 24 years. For each non-insulin antidiabetic drug, we calculated the annual prevalence proportion of users, overall and for specific age groups. From 2010 to 2019, the prevalence of non-insulin antidiabetic users in Scandinavia increased 37% from 0.43 to 0.59/1000 individuals. The prevalence proportions were highest among female adolescents and young adults, but the largest relative increase in use was seen among 10-14-year-olds (78%). Metformin was by far the most widely used non-insulin antidiabetic drug with a prevalence proportion of 0.51/1000 in 2019, followed by glucagon-like peptide-1 (GLP-1) analogues, which, however, showed an eight-fold relative increase during the study period.

The first junior European Calcium Society meeting: calcium research across scales, Kingdoms and countries.

The first junior European Calcium Society online meeting, held October 20-21, 2020, aimed to promote junior researchers in the Ca2+ community. The meeting included four scientific sessions, covering Ca2+ research from molecular detail to whole organisms. Each session featured one invited speaker and three speakers selected based on submitted abstracts, with the overall aim of actively involving early-career researchers. Consequently, the meeting underlined the diversity of Ca2+ physiology, by showcasing research across scales and Kingdoms, as presented by a correspondingly diverse speaker panel across career stages and countries. In this meeting report, we introduce the visions of the junior European Calcium Society board and summarize the meeting content.

Infanticide vs. inherited cardiac arrhythmias.

AIMS: In 2003, an Australian woman was convicted by a jury of smothering and killing her four children over a 10-year period. Each child died suddenly and unexpectedly during a sleep period, at ages ranging from 19 days to 18 months. In 2019 we were asked to investigate if a genetic cause could explain the children's deaths as part of an inquiry into the mother's convictions. METHODS AND RESULTS: Whole genomes or exomes of the mother and her four children were sequenced. Functional analysis of a novel CALM2 variant was performed by measuring Ca2+-binding affinity, interaction with calcium channels and channel function. We found two children had a novel calmodulin variant (CALM2 G114R) that was inherited maternally. Three genes (CALM1-3) encode identical calmodulin proteins. A variant in the corresponding residue of CALM3 (G114W) was recently reported in a child who died suddenly at age 4 and a sibling who suffered a cardiac arrest at age 5. We show that CALM2 G114R impairs calmodulin's ability to bind calcium and regulate two pivotal calcium channels (CaV1.2 and RyR2) involved in cardiac excitation contraction coupling. The deleterious effects of G114R are similar to those produced by G114W and N98S, which are considered arrhythmogenic and cause sudden cardiac death in children. CONCLUSION: A novel functional calmodulin variant (G114R) predicted to cause idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, or mild long QT syndrome was present in two children. A fatal arrhythmic event may have been triggered by their intercurrent infections. Thus, calmodulinopathy emerges as a reasonable explanation for a natural cause of their deaths.

Quantum dots-ßcyclodextrin-histidine labeled human adipose stem cells-laden chitosan hydrogel for bone tissue engineering.

Mesenchymal stem cells with differentiation ability to diverse cells play a crucial role in tissue engineering. Tracking the fate of these cells during the regeneration of tissue helps to obtain more information about their function. In this study, histidine conjugated ß-cyclodextrin as a cell-penetrating carrier with drug loading ability was attached to QDs nanoparticle (QD-ßCD-His) for stem cell labeling. Traceability of QD-ßCD-His labeled human adipose stem cells (hASCs) was monitored in 2D cell culture and 3D temperature-sensitive chitosan hydrogel scaffold. Dexamethasone (Dex) as an osteoinductive drug was loaded into QD-ßCD-His nano-carrier (QD-ßCD-His@Dex) to induce bone differentiation of labeled cells. Overall results indicated that QD-ßCD-His@Dex is a promising dual-purpose nano-carrier for stem cell labeling with osteoinductive potential in cell therapy as well as tissue engineering scaffolds.

Automated pupillometry and the FOUR score - what is the diagnostic benefit in neurointensive care?

INTRODUCTION: The Glasgow Coma Scale (GCS) and visual inspection of pupillary function are routine measures to monitor patients with impaired consciousness and predict their outcome in the neurointensive care unit (neuro-ICU). Our aim was to compare more recent measures, i.e. FOUR score and automated pupillometry, to standard monitoring with the GCS and visual inspection of pupils. METHODS: Supervised trained nursing staff examined a consecutive sample of patients admitted to the neuro-ICU of a tertiary referral centre using GCS and FOUR score and assessing pupillary function first by visual inspection and then by automated pupillometry. Clinical outcome was evaluated 6 months after admission using the Glasgow Outcome Scale-Extended. RESULTS: Fifty-six consecutive patients (median age 63 years) were assessed a total of 234 times. Of the 36 patients with at least one GCS score of 3, 13 had a favourable outcome. All seven patients with at least one FOUR score of ≤ 3 had an unfavourable outcome, which was best predicted by a low "brainstem" sub-score. Compared to automated pupillometry, visual assessment underestimated pupillary diameters (median difference, 0.4 mm; P = 0.006). Automated pupillometry detected a preserved pupillary light reflex in 10 patients, in whom visual inspection had missed pupillary constriction. DISCUSSION: Training of nursing staff to implement frequent monitoring of patients in the neuro-ICU with FOUR score and automated pupillometry is feasible. Both measures provide additional clinical information compared to the GCS and visual assessment of pupillary function, most importantly a more granular classification of patients with low levels of consciousness by the FOUR score.

New-Onset Atrial Fibrillation Among Patients With Infection in the Emergency Department: A Multicenter Cohort Study of 1-Year Stroke Risk.

BACKGROUND: Patients with new-onset atrial fibrillation in relation to infection are frequent in emergency departments (EDs) and may require antithrombotic therapy because of the increased risk of stroke. Our objective was to describe the 1-year risk of stroke in patients in the ED with infection, new-onset atrial fibrillation, and no antithrombotic therapy. METHODS: This was a population-based cohort study at 4 EDs in Denmark and Sweden. Atrial fibrillation was identified by electrocardiogram (ECG) upon arrival at the ED, and infection was identified by discharge diagnosis. Patient history was followed for 12 months or until initiation of oral anticoagulant therapy, ischemic stroke, or death. Primary outcome was stroke within 12 months compared to patients with infection and no atrial fibrillation. RESULTS: In the analysis, 15,505 patients were included; 48.7% were male and the median age was 71 (IQR, 56-83). Among the included patients, 2107 (13.6%) had atrial fibrillation of any kind and 822 (39.0%) of these had new-onset atrial fibrillation with a median CHA2DS2-VASc score of 3 (IQR 2-4). New-onset atrial fibrillation during infection showed an absolute postdischarge 1-year risk of stroke of 2.7% (95% CI 1.6-4.2), corresponding to a crude hazard ratio (HR) of 1.4 (95% CI 0.9-2.3), a sex and age adjusted HR of 1.0 (95% CI 0.6-1.6), and a CHA2DS2-VASc adjusted HR of 1.1 (95% CI, 0.7-1.8) compared to patients with infection but no atrial fibrillation. CONCLUSIONS: Patients in the ED with infection and new-onset atrial fibrillation without current oral anticoagulant therapy had a 2.7% absolute 1-year risk of stroke. Stroke events were mainly related to sex and age and risk factors identified by the CHA2DS2-VASc score.

Aquaporins differentially regulate cell-cell adhesion in MDCK cells.

Plasticity of epithelial cell-cell adhesion is vital in epithelial homeostasis and is regulated in multiple processes associated with cell migration, such as embryogenesis and wound healing. In cancer, cell-cell adhesion is compromised and is associated with increased cell migration and metastasis. Aquaporin (AQP) water channels facilitate water transport across cell membranes and are essential in the regulation of body water homeostasis. Increased expression of several AQPs, especially AQP5, is associated with increased cancer cell migration, metastasis, and poor prognosis. We found that AQP5 overexpression in normal epithelial cells induced cell detachment and dissemination from migrating cell sheets. AQP5 reduced both cell-cell coordination during collective migration and overall distance covered by the migrating cell sheets. AQP5 and the isoforms AQP1 and AQP4 decreased, whereas AQP3 increased, levels of plasma membrane-associated lateral junctional proteins. This regulation was mediated by the cytoplasmic domains of the AQPs. This shows that the AQPs have dual functions in epithelial physiology: as channel proteins and as differential regulators of cell-cell adhesiveness. This regulation may contribute to dynamic regulation of cell junctions in processes such as embryogenesis and wound healing and also explain the pivotal roles of AQPs in carcinogenesis and metastasis.-Login, F. H., Jensen, H. H., Pedersen, G. A., Koffman, J. S., Kwon, T.-H., Parsons, M., Nejsum, L. N. Aquaporins differentially regulate cell-cell adhesion in MDCK cells.

Etiology of Shock in the Emergency Department: A 12-Year Population-Based Cohort Study.

INTRODUCTION: The knowledge of the etiology and associated mortality of undifferentiated shock in the emergency department (ED) is limited. We aimed to describe the etiology-based proportions and incidence rates (IR) of shock, as well as the associated mortality in the ED. METHODS: Population-based cohort study at a University Hospital ED in Denmark from January 1, 2000, to December 31, 2011. Patients aged ≥18 years living in the ED-catchment area (N = 225,000) with a first-time ED presentation with shock (n = 1,553) defined as hypotension (systolic blood pressure ≤100 mm Hg) and ≥1 organ failures were included. Discharge diagnoses defined the etiology and were grouped as follows: distributive septic shock (SS), distributive non-septic shock (NS), cardiogenic shock (CS), hypovolemic shock (HS), obstructive shock (OS), and other conditions (OC). Outcomes were etiology-based characteristics, annual IR per 100,000 person-years at risk (95% confidence intervals [CIs]), mortality at 0 to 7-, and 0 to 90 days (95% CIs) and hazard rates (HR) at 0 to 7, 8 to 90 days (95% CIs). Poisson and Cox regression models were used for analyses. RESULTS: Among 1,553 shock patients: 423 (27.2%) had SS, 363 (23.4%) NS, 217 (14.0%) CS, 479 (30.8%) HS, 14 (0.9%) OS, and 57 (3.7%) OC. The corresponding IRs were 16.2/100,000 (95% CI: 14.8-17.9), 13.9/100,000 (95% CI: 12.6-15.4), 8.3/100,000 (95% CI: 7.3-9.5), 18.4/100,000 (95% CI: 16.8-20.1), 0.5/100,000 (95% CI: 0.3-0.9), and 2.2/100,000 (95% CI: 1.7-2.8). SS IR increased from 8.4 to 28.5/100,000 during the period 2000 to 2011. Accordingly, the 7-, and 90-day mortalities of SS, NS, CS, and HS were 30.3% (95% CI: 25.9-34.7) and 56.2% (95% CI: 50.7-61.5), 12.7% (95% CI: 9.2-16.1) and 22.6% (95% CI: 18.1-27.7), 34.6% (95% CI: 28.2-40.9) and 52.3% (95% CI: 44.6-59.8), 19.2% (95% CI: 15.7-22.7), and 36.8% (95% CI: 33.3-43.3). SS (HR = 1.46 [95% CI: 1.03-2.07]), and CS (HR = 2.15 [95% CI: 1.47-3.13]) were independent predictors of death within 0 to 7 days, whereas SS was a predictor within 8 to 90 days (HR = 1.66 [95% CI: 1.14-2.42]). CONCLUSION: HS and SS are frequent etiological characteristics followed by NS and CS, whereas OS is a rare condition. We confirm the increasing trend of SS, as previously reported. Seven-day mortality ranged from 12.7% to 34.6%, while 90-day mortality ranged from 22.6% to 56.2%. The underlying etiology was an independent predictor of mortality.

The Na+ /H+ exchanger NHE1 localizes as clusters to cryptic lamellipodia and accelerates collective epithelial cell migration.

KEY POINTS: Exogenous Na+ /H+ exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets Stimulation with epidermal growth factor, a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows NHE1 expression disrupted the morphology of epithelial cell sheets and three-dimensional cysts ABSTRACT: Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na+ /H+ exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, although its possible role in collective migration of normal epithelial cells has remained unresolved. In the present study, we show that NHE1 expression in MDCK-II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor, a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased the number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and epidermal growth factor. Finally, NHE1 expression resulted in disorganized development of MDCK-II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development.

Regulation of Plasma Membrane Nanodomains of the Water Channel Aquaporin-3 Revealed by Fixed and Live Photoactivated Localization Microscopy.

Several aquaporin (AQP) water channels are short-term regulated by the messenger cyclic adenosine monophosphate (cAMP), including AQP3. Bulk measurements show that cAMP can change diffusive properties of AQP3; however, it remains unknown how elevated cAMP affects AQP3 organization at the nanoscale. Here we analyzed AQP3 nano-organization following cAMP stimulation using photoactivated localization microscopy (PALM) of fixed cells combined with pair correlation analysis. Moreover, in live cells, we combined PALM acquisitions of single fluorophores with single-particle tracking (spt-PALM). These analyses revealed that AQP3 tends to cluster and that the diffusive mobility is confined to nanodomains with radii of ∼150 nm. This domain size increases by ∼30% upon elevation of cAMP, which, however, is not accompanied by a significant increase in the confined diffusion coefficient. This regulation of AQP3 organization at the nanoscale may be important for understanding the mechanisms of water AQP3-mediated water transport across plasma membranes.

Human Calmodulin Mutations.

Fluxes of calcium (Ca2+) across cell membranes enable fast cellular responses. Calmodulin (CaM) senses local changes in Ca2+ concentration and relays the information to numerous interaction partners. The critical role of accurate Ca2+ signaling on cellular function is underscored by the fact that there are three independent CaM genes (CALM1-3) in the human genome. All three genes are functional and encode the exact same CaM protein. Moreover, CaM has a completely conserved amino acid sequence across all vertebrates. Given this degree of conservation, it was long thought that mutations in CaM were incompatible with life. It was therefore a big surprise when the first CaM mutations in humans were identified six years ago. Today, more than a dozen human CaM missense mutations have been described, all found in patients with severe cardiac arrhythmias. Biochemical studies have demonstrated differential effects on Ca2+ binding affinities for these CaM variants. Moreover, CaM regulation of central cardiac ion channels is impaired, including the voltage-gated Ca2+ channel, CaV1.2, and the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor isoform 2, RyR2. Currently, no non-cardiac phenotypes have been described for CaM variant carriers. However, sequencing of large human cohorts reveals a cumulative frequency of additional rare CaM mutations that raise the possibility of CaM variants not exclusively causing severe cardiac arrhythmias. Here, we provide an overview of the identified CaM variants and their known consequences for target regulation and cardiac disease phenotype. We discuss experimental data, patient genotypes and phenotypes as well as which questions remain open to understand this complexity.