Effects of interventions targeting the systemic inflammatory response to cardiac surgery on clinical outcomes in adults.

BACKGROUND: Organ injury is a common and severe complication of cardiac surgery that contributes to the majority of deaths. There are no effective treatment or prevention strategies. It has been suggested that innate immune system activation may have a causal role in organ injury. A wide range of organ protection interventions targeting the innate immune response have been evaluated in randomised controlled trials (RCTs) in adult cardiac surgery patients, with inconsistent results in terms of effectiveness. OBJECTIVES: The aim of the review was to summarise the results of RCTs of organ protection interventions targeting the innate immune response in adult cardiac surgery. The review considered whether the interventions had a treatment effect on inflammation, important clinical outcomes, or both. SEARCH METHODS: CENTRAL, MEDLINE, Embase, conference proceedings and two trial registers were searched on October 2022 together with reference checking to identify additional studies. SELECTION CRITERIA: RCTs comparing organ protection interventions targeting the innate immune response versus placebo or no treatment in adult patients undergoing cardiac surgery where the treatment effect on innate immune activation and on clinical outcomes of interest were reported. DATA COLLECTION AND ANALYSIS: Searches, study selection, quality assessment, and data extractions were performed independently by pairs of authors. The primary inflammation outcomes were peak IL-6 and IL-8 concentrations in blood post-surgery. The primary clinical outcome was in-hospital or 30-day mortality. Treatment effects were expressed as risk ratios (RR) and standardised mean difference (SMD) with 95% confidence intervals (CI). Meta-analyses were performed using random effects models, and heterogeneity was assessed using I2. MAIN RESULTS: A total of 40,255 participants from 328 RCTs were included in the synthesis. The effects of treatments on IL-6 (SMD -0.77, 95% CI -0.97 to -0.58, I2 = 92%) and IL-8 (SMD -0.92, 95% CI -1.20 to -0.65, I2 = 91%) were unclear due to heterogeneity. Heterogeneity for inflammation outcomes persisted across multiple sensitivity and moderator analyses. The pooled treatment effect for in-hospital or 30-day mortality was RR 0.78, 95% CI 0.68 to 0.91, I2 = 0%, suggesting a significant clinical benefit. There was little or no treatment effect on mortality when analyses were restricted to studies at low risk of bias. Post hoc analyses failed to demonstrate consistent treatment effects on inflammation and clinical outcomes. Levels of certainty for pooled treatment effects on the primary outcomes were very low. AUTHORS' CONCLUSIONS: A systematic review of RCTs of organ protection interventions targeting innate immune system activation did not resolve uncertainty as to the effectiveness of these treatments, or the role of innate immunity in organ injury following cardiac surgery.

Urinary extracellular vesicles and micro-RNA as markers of acute kidney injury after cardiac surgery.

We hypothesised that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. The study was performed in the cohort of the REVAKI-2 trial. Urine samples were collected before and 24 h after the procedure from 94 cardiac surgery patients. Urinary particle concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced, and verification was performed with advanced TaqMan assays in samples from all patients. Urinary particle concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for CD105 and ß1-integrin also increasing. Pre-surgery podocalyxin-positive EV were significantly lower in patients with AKI. Their levels correlated with the severity of the injury. Pre-operative miR-125a-5p was expressed at lower levels in urine from patients with AKI when adjusted for urinary creatinine. Levels of miR-10a-5p were lower after surgery in AKI patients and its levels correlated with the severity of the injury. Pre-operative levels of podocalyxin EVs, urinary particle concentrations and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 82%) AKI prediction.

A systematic review of micro-RNAs in aortic stenosis and cardiac fibrosis.

Aortic stenosis (AS) is the commonest valve lesion requiring surgery in the Western world. The presence of myocardial fibrosis is associated with mortality even after valve replacement. MicroRNAs could serve as biomarkers of fibrosis and risk stratify patients for earlier intervention. This study aimed to systematically review reports of micro-RNA (miR) associated with fibrosis in AS and identify potential biomarkers. We searched EMBASE, Medline, and Web of Science up to May 2020. Studies that reported on the role of miRs in AS and cardiac fibrosis were included. Study quality was assessed using the Newcastle-Ottawa scale. Of 4230 reports screened, 25 were included. All studies were of low to moderate quality. MiRs were analyzed in myocardial tissue (n = 10), aortic valve tissue (n = 5), plasma (n = 5), and serum (n = 5). A total of 365 miRs were reported, of which only a few were reported in more than one paper (3 in the myocardium, 5 in the aortic valve, and 1 in plasma). miR-21 was upregulated in plasma and myocardial tissue. MiR-19b was downregulated in the myocardium. Papers reporting myocardial miR-1 contradicted each other, and miR-133a was associated with increased left ventricular mass regression post-surgery. In the aortic valve, miRs-665, 602 and 939 were downregulated, and miRs-193b and 214 were upregulated. The data on miR in fibrosis in AS is scarce and of low to moderate quality. Further studies are needed to identify novel miRs as biomarkers, especially at an earlier asymptomatic phase of the disease.

Gene and metabolite expression dependence on body mass index in human myocardium.

We hypothesized that body mass index (BMI) dependent changes in myocardial gene expression and energy-related metabolites underlie the biphasic association between BMI and mortality (the obesity paradox) in cardiac surgery. We performed transcriptome profiling and measured a panel of 144 metabolites in 53 and 55, respectively, myocardial biopsies from a cohort of sixty-six adult patients undergoing coronary artery bypass grafting (registration: NCT02908009). The initial analysis identified 239 transcripts with biphasic BMI dependence. 120 displayed u-shape and 119 n-shape expression patterns. The identified local minima or maxima peaked at BMI 28-29. Based on these results and to best fit the WHO classification, we grouped the patients into three groups: BMI < 25, 25 ≤ BMI ≤ 32, and BMI > 32. The analysis indicated that protein translation-related pathways were downregulated in 25 ≤ BMI ≤ 32 compared with BMI < 25 patients. Muscle contraction transcripts were upregulated in 25 ≤ BMI ≤ 32 patients, and cholesterol synthesis and innate immunity transcripts were upregulated in the BMI > 32 group. Transcripts involved in translation, muscle contraction and lipid metabolism also formed distinct correlation networks with biphasic dependence on BMI. Metabolite analysis identified acylcarnitines and ribose-5-phosphate increasing in the BMI > 32 group and α-ketoglutarate increasing in the BMI < 25 group. Molecular differences in the myocardium mirror the biphasic relationship between BMI and mortality.

Activation of the innate immune response and organ injury after cardiac surgery: a systematic review and meta-analysis of randomised trials and analysis of individual patient data from randomised and non-randomised studies.

BACKGROUND: It is unclear whether the innate immune response represents a therapeutic target for organ protection strategies in cardiac surgery. METHODS: A systematic review of trials of interventions targeting the inflammatory response to cardiac surgery reporting treatment effects on both innate immune system cytokines and organ injury was performed. The protocol was registered at the International Prospective Register of Systematic Reviews: CRD42020187239. Searches of the Cochrane Central Register of Controlled Trials, MEDLINE, and Embase were performed. Random-effects meta-analyses were used for the primary analysis. A separate analysis of individual patient data from six studies (n=785) explored sources of heterogeneity for treatment effects on cytokine levels. RESULTS: Searches to May 2020 identified 251 trials evaluating 24 interventions with 20 582 participants for inclusion. Most trials had important limitations. Methodological limitations of the included trials and heterogeneity of the treatment effects on cytokine levels between trials limited interpretation. The primary analysis demonstrated inconsistency in the direction of the treatment effects on innate immunity and organ failure or death between interventions. Analyses restricted to important subgroups or trials with fewer limitations showed similar results. Meta-regression, pooling available data from all trials, demonstrated no association between the direction of the treatment effects on inflammatory cytokines and organ injury or death. The analysis of individual patient data demonstrated heterogeneity in the association between the cytokine response and organ injury after cardiac surgery for people >75 yr old and those with some chronic diseases. CONCLUSIONS: The certainty of the evidence for a causal relationship between innate immune system activation and organ injury after cardiac surgery is low.

Systematic Review and Meta-Analysis of Diagnostic Test Accuracy Studies Evaluating Point-of-Care Tests of Coagulopathy in Cardiac Surgery.

Treatment guidelines recommend the routine use of point-of-care diagnostic tests for coagulopathy in the management of cardiac surgery patients at risk of severe bleeding despite uncertainty as to their diagnostic accuracy. We performed a systematic review and meta-analysis of studies that evaluated the diagnostic accuracy of viscoelastometry, platelet function tests, and modified thromboelastography (TEG) tests, for coagulopathy in cardiac surgery patients. The reference standard included resternotomy for bleeding, transfusion of non-red cell components, or massive transfusion. We searched MEDLINE, EMBASE, CINAHL, and Clinical Trials.gov, from inception to June 2019. Study quality was assessed using QUADAS-2. Bivariate models were used to estimate summary sensitivity and specificity with (95% confidence intervals). All 29 studies (7440 participants) included in the data synthesis evaluated the tests as predictors of bleeding. No study evaluated their role in the management of bleeding. None was at low risk of bias. Four were judged as low concern regarding applicability. Pooled estimates of diagnostic accuracy were; Viscoelastic tests, 12 studies, sensitivity 0.61 (0.44, 0.76), specificity 0.83 (0.70, 0.91) with significant heterogeneity. Platelet function tests, 12 studies, sensitivity 0.63 (0.53, 0.72), specificity 0.75 (0.64, 0.84) with significant heterogeneity. TEG modification tests, 3 studies, sensitivity 0.80 (0.67, 0.89), specificity 0.76 (0.69, 0.82) with no evidence of heterogeneity. Studies reporting the highest values for sensitivity and specificity had important methodological limitations. In conclusion, we did not demonstrate predictive accuracy for commonly used point-of-care devices for coagulopathic bleeding in cardiac surgery. However, the certainty of the evidence was low.

Biomarkers of Inflammation and Lung Recovery in Extracorporeal Membrane Oxygenation Patients With Persistent Pulmonary Hypertension of the Newborn: A Feasibility Study.

OBJECTIVES: Extracorporeal membrane oxygenation is a treatment for Persistent Pulmonary Hypertension of the Newborn with high mortality. HYPOTHESIS: the extracorporeal membrane oxygenation circuit results in inflammatory responses that mitigate against successful weaning. DESIGN: Single-center prospective observational feasibility study. SETTING: PICU. PATIENTS: Twenty-four neonates requiring extracorporeal membrane oxygenation support for Persistent Pulmonary Hypertension of the Newborn. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The reference outcome was death or more than 7 days of extracorporeal membrane oxygenation support. Other outcomes included serial measures of plasma-free hemoglobin and markers of its metabolism, leucocyte, platelet and endothelial activation, and biomarkers of inflammation. Of 24 participants recruited between February 2016 and June 2017, 10 died or required prolonged extracorporeal membrane oxygenation support. These patients were sicker at baseline with higher levels of plasma-free hemoglobin within 12 hours of cannulation (geometric mean ratio, 1.92; 95% CIs, 1.00-3.67; p = 0.050) but not thereafter, versus those requiring less than 7 days extracorporeal membrane oxygenation. Serum haptoglobin concentrations were significantly elevated in both groups. Patients who died or required prolonged extracorporeal membrane oxygenation support demonstrated elevated levels of platelet-leucocyte aggregation, but decreased concentrations of mediators of the inflammatory response: interleukin-8, C-reactive protein, and tumor necrosis factor α. CONCLUSIONS: Clinical status at baseline and not levels of plasma-free hemoglobin or the systemic inflammatory response may determine the requirement for prolonged extracorporeal membrane oxygenation support in neonates.

An Observational Cohort Feasibility Study to Identify Microvesicle and Micro-RNA Biomarkers of Acute Kidney Injury Following Pediatric Cardiac Surgery.

OBJECTIVES: Micro-RNA, small noncoding RNA fragments involved in gene regulation, and microvesicles, membrane-bound particles less than 1 µm known to regulate cellular processes including responses to injury, may serve as disease-specific biomarkers of acute kidney injury. We evaluated the feasibility of measuring these signals as well as other known acute kidney injury biomarkers in a mixed pediatric cardiac surgery population. DESIGN: Single center prospective cohort feasibility study. SETTING: PICU. PATIENTS: Twenty-four children (≤ 17 yr) undergoing cardiac surgery with cardiopulmonary bypass without preexisting inflammatory state, acute kidney injury, or extracorporeal life support. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Acute kidney injury was defined according to modified Kidney Diseases Improving Global Outcomes criteria. Blood and urine samples were collected preoperatively and at 6-12 and 24 hours. Microvesicles derivation was assessed using flow cytometry and NanoSight analysis. Micro-RNAs were isolated from plasma and analyzed by microarray and quantitative real-time polymerase chain reaction. Data completeness for the primary outcomes was 100%. Patients with acute kidney injury (n = 14/24) were younger, underwent longer cardiopulmonary bypass, and required greater inotrope support. Acute kidney injury subjects had different fractional content of platelets and endothelial-derived microvesicles before surgery. Platelets and endothelial microvesicles levels were higher in acute kidney injury patients. A number of micro-RNA species were differentially expressed in acute kidney injury patients. Pathway analysis of candidate target genes in the kidney suggested that the most often affected pathways were phosphatase and tensin homolog and signal transducer and activator of transcription 3 signaling. CONCLUSIONS: Microvesicles and micro-RNAs expression patterns in pediatric cardiac surgery patients can be measured in children and potentially serve as tools for stratification of patients at risk of acute kidney injury.

A Comparison of Red Cell Rejuvenation versus Mechanical Washing for the Prevention of Transfusion-associated Organ Injury in Swine.

BACKGROUND: We evaluated the effects of two interventions that modify the red cell storage lesion on kidney and lung injury in experimental models of transfusion. METHODS: White-landrace pigs (n = 32) were allocated to receive sham transfusion (crystalloid), 14-day stored allogeneic red cells, 14-day red cells washed using the red cells washing/salvage system (CATS; Fresenius, Germany), or 14-day red cells rejuvenated using the inosine solution (Rejuvesol solution; Zimmer Biomet, USA) and washed using the CATS device. Functional, biochemical, and histologic markers of organ injury were assessed for up to 24 h posttransfusion. RESULTS: Transfusion of 14 day red cells resulted in lung injury (lung injury score vs. sham, mean difference -0.3 (95% CI, -0.6 to -0.1; P = 0.02), pulmonary endothelial dysfunction, and tissue leukocyte sequestration. Mechanical washing reduced red cell-derived microvesicles but increased cell-free hemoglobin in 14-day red cell units. Transfusion of washed red cells reduced leukocyte sequestration but did not reduce the lung injury score (mean difference -0.2; 95% CI, -0.5 to 0.1; P = 0.19) relative to 14-day cells. Transfusion of washed red cells also increased endothelial activation and kidney injury. Rejuvenation restored adenosine triphosphate to that of fresh red cells and reduced microvesicle concentrations without increasing cell-free hemoglobin release. Transfusion of rejuvenated red cells reduced plasma cell-free hemoglobin, leukocyte sequestration, and endothelial dysfunction in recipients and reduced lung and kidney injury relative to 14-day or washed 14-day cells. CONCLUSIONS: Reversal of the red cell storage lesion by rejuvenation reduces transfusion-associated organ injury in swine.

Body Mass Index and Mortality Among Adults Undergoing Cardiac Surgery: A Nationwide Study With a Systematic Review and Meta-Analysis.

BACKGROUND: In an apparent paradox, morbidity and mortality are lower in obese patients undergoing cardiac surgery, although the nature of this association is unclear. We sought to determine whether the obesity paradox observed in cardiac surgery is attributable to reverse epidemiology, bias, or confounding. METHODS: Data from the National Adult Cardiac Surgery registry for all cardiac surgical procedures performed between April 2002 and March 2013 were extracted. A parallel systematic review and meta-analysis (MEDLINE, Embase, SCOPUS, Cochrane Library) through June 2015 were also accomplished. Exposure of interest was body mass index categorized into 6 groups according to the World Health Organization classification. RESULTS: A total of 401 227 adult patients in the cohort study and 557 720 patients in the systematic review were included. A U-shaped association between mortality and body mass index classes was observed in both studies, with lower mortality in overweight (adjusted odds ratio, 0.79; 95% confidence interval, 0.76-0.83) and obese class I and II (odds ratio, 0.81; 95% confidence interval, 0.76-0.86; and odds ratio, 0.83; 95% confidence interval, 0.74-0.94) patients relative to normal-weight patients and increased mortality in underweight individuals (odds ratio, 1.51; 95% confidence interval, 1.41-1.62). In the cohort study, a U-shaped relationship was observed for stroke and low cardiac output syndrome but not for renal replacement therapy or deep sternal wound infection. Counter to the reverse epidemiology hypotheses, the protective effects of obesity were less in patients with severe chronic renal, lung, or cardiac disease and greater in older patients and in those with complications of obesity, including the metabolic syndrome and atherosclerosis. Adjustments for important confounders did not alter our results. CONCLUSIONS: Obesity is associated with lower risks after cardiac surgery, with consistent effects noted in multiple analyses attempting to address residual confounding and reverse causation.

Tumour Suppressor Adenomatous Polyposis Coli (APC) localisation is regulated by both Kinesin-1 and Kinesin-2.

Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.

Further observations on the clinical significance and inheritance of the low-frequency platelet antigen HPA-28bw.

BACKGROUND: Most recently described human platelet antigens (HPAs) have been low-frequency polymorphisms identified in cases of fetomaternal alloimmune thrombocytopenia (FMAIT). There is limited opportunity to study the clinical significance or different antenatal management strategies in cases involving low-frequency HPA antibodies because many are single pregnancies. We have previously described a low-frequency platelet (PLT) antigen, HPA-28bw, implicated in FMAIT in two of the three infants in the same family. This report describes the outcome of an additional two pregnancies in this family. STUDY DESIGN AND METHODS: The fourth and fifth pregnancies in a HPA-28bw-alloimmunized mother with a heterozygous partner were investigated to determine the risk of FMAIT. The presence of anti-HPA-28bw was assessed by paternal crossmatch studies. Prenatal HPA genotyping of amniocytes was performed to inform antenatal management. RESULTS: GPIIb/IIIa antibodies reactive only with paternal PLTs were detected. These antibodies had been previously identified as HPA-28bw specific using recombinant GPIIb glycoprotein mutated to contain the HPA-28bw (V740L) mutation. The fetus in the fourth pregnancy did not inherit the HPA-28bw mutation, no antenatal management was required, and the baby had a normal PLT count. The fetus in the fifth pregnancy did inherit the HPA-28bw mutation. The mother received IVIG (2 g/kg/week) and prednisolone during pregnancy, and the baby was born with a normal PLT count. CONCLUSION: Study of this family has provided a unique opportunity to assess the clinical significance of antibodies against the low-frequency PLT antigen (HPA-28bw) during five pregnancies and to compare the outcomes of different antenatal treatments.

A V740L mutation in glycoprotein IIb defines a novel epitope (War) associated with fetomaternal alloimmune thrombocytopenia.

BACKGROUND: Most recently described human platelet antigens (HPAs) have been detected in cases of fetomaternal alloimmune thrombocytopenia (FMAIT) where the mother has been immunized against a low-frequency antigen that the fetus has inherited from the father. Low-frequency antigens are not represented in normal panel platelets (PLTs) and antibody detection and identification in such cases requires incubation of maternal serum with paternal PLTs and definition of the causative mutation. STUDY DESIGN AND METHODS: A suspected case of FMAIT was investigated for PLT-specific antibodies using a panel of both HPA-typed and paternal PLTs. HPA typing was performed by polymerase chain reaction with sequence-specific primers and further DNA analysis was performed using direct sequencing of the coding regions of the ITGA2B and ITGB3 genes. RESULTS: Maternal antibodies reactive only with paternal PLTs were localized to glycoprotein (GP)IIb/IIIa using the monoclonal antibody immobilization of PLT antibody assay. A single-nucleotide polymorphism was detected in Exon 23 of ITGA2B in the father and affected child, which predicted a V740L substitution in the mature protein. Recombinant V740L mutated GPIIb expressed in HEK293 cells was specifically recognized by maternal antibodies. The polymorphism was not detected either in the mother or in a cohort of 100 donors. CONCLUSION: The V740L polymorphism defines a new low-frequency antigen implicated in two cases of FMAIT in a single family. Low-frequency HPAs are clinically important and their elucidation requires both crossmatch studies and gene sequencing in cases where there is strong clinical evidence of FMAIT but initial laboratory investigations do not support the diagnosis.

Detection of HNA-3a and -3b antibodies using transfected cell lines and recombinant proteins.

BACKGROUND: HNA-3 is a diallellic system located on choline transporter-like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA-3a antibodies are of clinical importance in transfusion-related acute lung injury but antibody detection requires labor-intensive granulocyte isolation from HNA-typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55-230) and full-length membrane bound CTL2 with HNA-3a and -3b epitopes. Soluble fragments were used in enzyme-linked immunosorbent assays to detect HNA-3 antibodies. The cell lines expressing full-length proteins were trypsin treated to remove HLA antigens and frozen at -80°C. Thawed cells were then used to detect HNA-3 antibodies by flow cytometry. RESULTS: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti-HNA-3a sera and by both available anti-HNA-3b sera. Twenty-one anti-HLA sera reacted variably with untreated cell lines expressing full-length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti-HNA-3a and two anti-HNA-3b sera bound to the corresponding cell line. CONCLUSION: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA-3 antibodies, the HEK293 cells expressing full-length CTL2 proteins were useful in the detection of HNA-3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use.

Role of kinesin-1 and cytoplasmic dynein in endoplasmic reticulum movement in VERO cells.

Generating the extended endoplasmic reticulum (ER) network depends on microtubules, which act as tracks for motor-driven ER tubule movement, generate the force to extend ER tubules by means of attachment to growing microtubule plus-ends and provide static attachment points. We have analysed ER dynamics in living VERO cells and find that most ER tubule extension is driven by microtubule motors. Surprisingly, we observe that approximately 50% of rapid ER tubule movements occur in the direction of the centre of the cell, driven by cytoplasmic dynein. Inhibition of this movement leads to an accumulation of lamellar ER in the cell periphery. By expressing dominant-negative kinesin-1 constructs, we show that kinesin-1 drives ER tubule extension towards the cell periphery and that this motility is dependent on the KLC1B kinesin light chain splice form but not on KLC1D. Inhibition of kinesin-1 promotes a shift from tubular to lamellar morphology and slows down the recovery of the ER network after microtubule depolymerisation and regrowth. These observations reconcile previous conflicting studies of kinesin-1 function in ER motility in vivo. Furthermore, our data reveal that cytoplasmic dynein plays a role in ER motility in a mammalian cultured cell, demonstrating that ER motility is more complex than previously thought.

Cargo selection by specific kinesin light chain 1 isoforms.

Kinesin-1 drives the movement of diverse cargoes, and it has been proposed that specific kinesin light chain (KLC) isoforms target kinesin-1 to these different structures. Here, we test this hypothesis using two in vitro motility assays, which reconstitute the movement of rough endoplasmic reticulum (RER) and vesicles present in a Golgi membrane fraction. We generated GST-tagged fusion proteins of KLC1B and KLC1D that included the tetratricopeptide repeat domain and the variable C-terminus. We find that preincubation of RER with KLC1B inhibits RER motility, whereas KLC1D does not. In contrast, Golgi fraction vesicle movement is inhibited by KLC1D but not KLC1B reagents. Both RER and vesicle movement is inhibited by preincubation with the GST-tagged C-terminal domain of ubiquitous kinesin heavy chain (uKHC), which binds to the N-terminal domain of uKHC and alters its interaction with microtubules. We propose that although the TRR domains are required for cargo binding, it is the variable C-terminal region of KLCs that are vital for targeting kinesin-1 to different cellular structures.

The novel protein KBP regulates mitochondria localization by interaction with a kinesin-like protein.

BACKGROUND: Members of the Kinesin-3 family of kinesin-like proteins mediate transport of axonal vesicles (KIF1A, KIF1Bbeta), distribution of mitochondria (KIF1Balpha) and anterograde Golgi to ER vesicle transport (KIF1C). Until now, little is known about the regulation of kinesin-like proteins. Several proteins interact with members of this protein family. Here we report on a novel, KIF1 binding protein (KBP) that was identified in yeast two-hybrid screens. RESULTS: KBP was identified by using the yeast-two-hybrid system with an amino-terminal fragment of KIF1C as a bait that is strongly homologous to KIF1B. Here we investigated the interaction of KBP and KIF1B. The full length proteins coimmunoprecipitated after overexpression and in untransfected 293 cells. Immunofluorescence experiments revealed that KBP was mainly localized to mitochondria, as has been described for KIF1Balpha. Overexpression of a deletion mutant or reduction of the KBP protein level using an anti-sense construct led to an aggregation of mitochondria. Such an effect is probably due to the lower activity of KIF1Balpha in the absence of KBP, as was revealed in motility assays. CONCLUSION: KBP is a new binding partner for KIF1Balpha that is a regulator of its transport function and thus represents a new type of kinesin interacting protein.

Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint.

Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.

N-terminal kinesins: many and various.

Molecular motors are a fascinating group of proteins that have vital roles in a huge variety of cellular processes. They all share the ability to produce force through the hydrolysis of adenosine triphosphate, and fall into classes groups: the kinesins, myosins and the dyneins. The kinesin superfamily itself can be split into three major groups depending on the position of the motor domain, which is localized N-terminally, C-terminally, or internally. This review focuses on the N-terminal kinesins, providing a brief overview of their roles within the cell, and illustrating recent key developments in our understanding of how these proteins function.