Assessing prospective and retrospective metacognitive accuracy following traumatic brain injury remotely across cognitive domains.

The ability to monitor one's behaviour is frequently impaired following TBI, impacting on patients' rehabilitation. Inaccuracies in judgement or self-reflection of one's performance provides a useful marker of metacognition. However, metacognition is rarely measured during routine neuropsychology assessments and how it varies across cognitive domains is unclear. A cohort of participants consisting of 111 TBI patients [mean age = 45.32(14.15), female = 29] and 84 controls [mean age = 31.51(12.27), female = 43] was studied. Participants completed cognitive assessments via a bespoke digital platform on their smartphones. Included in the assessment were a prospective evaluation of memory and attention, and retrospective confidence judgements of task performance. Metacognitive accuracy was calculated from the difference between confidence judgement of task performance and actual performance. Prospective judgment of attention and memory was correlated with task performance in these domains for controls but not patients. TBI patients had lower task performance in processing speed, executive functioning and working memory compared to controls, maintaining high confidence, resulting in overestimation of cognitive performance compared to controls. Additional judgments of task performance complement neuropsychological assessments with little additional time-cost. These results have important theoretical and practical implications for evaluation of metacognitive impairment in TBI patients and neurorehabilitation.

Brain age predicts mortality.

Age-associated disease and disability are placing a growing burden on society. However, ageing does not affect people uniformly. Hence, markers of the underlying biological ageing process are needed to help identify people at increased risk of age-associated physical and cognitive impairments and ultimately, death. Here, we present such a biomarker, 'brain-predicted age', derived using structural neuroimaging. Brain-predicted age was calculated using machine-learning analysis, trained on neuroimaging data from a large healthy reference sample (N=2001), then tested in the Lothian Birth Cohort 1936 (N=669), to determine relationships with age-associated functional measures and mortality. Having a brain-predicted age indicative of an older-appearing brain was associated with: weaker grip strength, poorer lung function, slower walking speed, lower fluid intelligence, higher allostatic load and increased mortality risk. Furthermore, while combining brain-predicted age with grey matter and cerebrospinal fluid volumes (themselves strong predictors) not did improve mortality risk prediction, the combination of brain-predicted age and DNA-methylation-predicted age did. This indicates that neuroimaging and epigenetics measures of ageing can provide complementary data regarding health outcomes. Our study introduces a clinically-relevant neuroimaging ageing biomarker and demonstrates that combining distinct measurements of biological ageing further helps to determine risk of age-related deterioration and death.

Relationships between the integrity and function of lumbar nerve roots as assessed by diffusion tensor imaging and neurophysiology.

PURPOSE: Diffusion tensor imaging (DTI) has shown promise in the measurement of peripheral nerve integrity, although the optimal way to apply the technique for the study of lumbar spinal nerves is unclear. The aims of this study are to use an improved DTI acquisition to investigate lumbar nerve root integrity and correlate this with functional measures using neurophysiology. METHODS: Twenty healthy volunteers underwent 3 T DTI of the L5/S1 area. Regions of interest were applied to L5 and S1 nerve roots, and DTI metrics (fractional anisotropy, mean, axial and radial diffusivity) were derived. Neurophysiological measures were obtained from muscles innervated by L5/S1 nerves; these included the slope of motor-evoked potential input-output curves, F-wave latency, maximal motor response, and central and peripheral motor conduction times. RESULTS: DTI metrics were similar between the left and right sides and between vertebral levels. Conversely, significant differences in DTI measures were seen along the course of the nerves. Regression analyses revealed that DTI metrics of the L5 nerve correlated with neurophysiological measures from the muscle innervated by it. CONCLUSION: The current findings suggest that DTI has the potential to be used for assessing lumbar spinal nerve integrity and that parameters derived from DTI provide quantitative information which reflects their function.

A pragmatic randomized controlled trial to evaluate the effectiveness of a facilitated exercise intervention as a treatment for postnatal depression: the PAM-PeRS trial.

BACKGROUND: Postnatal depression affects about 10-15% of women in the year after giving birth. Many women and healthcare professionals would like an effective and accessible non-pharmacological treatment for postnatal depression. METHOD: Women who fulfilled the International Classification of Diseases (ICD)-10 criteria for major depression in the first 6 months postnatally were randomized to receive usual care plus a facilitated exercise intervention or usual care only. The intervention involved two face-to-face consultations and two telephone support calls with a physical activity facilitator over 6 months to support participants to engage in regular exercise. The primary outcome was symptoms of depression using the Edinburgh Postnatal Depression Scale (EPDS) at 6 months post-randomization. Secondary outcomes included EPDS score as a binary variable (recovered and improved) at 6 and 12 months post-randomization. RESULTS: A total of 146 women were potentially eligible and 94 were randomized. Of these, 34% reported thoughts of self-harming at baseline. After adjusting for baseline EPDS, analyses revealed a -2.04 mean difference in EPDS score, favouring the exercise group [95% confidence interval (CI) -4.11 to 0.03, p = 0.05]. When also adjusting for pre-specified demographic variables the effect was larger and statistically significant (mean difference = -2.26, 95% CI -4.36 to -0.16, p = 0.03). Based on EPDS score a larger proportion of the intervention group was recovered (46.5% v. 23.8%, p = 0.03) compared with usual care at 6 months follow-up. CONCLUSIONS: This trial shows that an exercise intervention that involved encouragement to exercise and to seek out social support to exercise may be an effective treatment for women with postnatal depression, including those with thoughts of self-harming.

Sedentary time and markers of inflammation in people with newly diagnosed type 2 diabetes.

BACKGROUND AND AIMS: We investigated whether objectively measured sedentary time was associated with markers of inflammation in adults with newly diagnosed type 2 diabetes. METHODS AND RESULTS: We studied 285 adults (184 men, 101 women, mean age 59.0 ± 9.7) who had been recruited to the Early ACTivity in Diabetes (Early ACTID) randomised controlled trial. C-reactive protein (CRP), adiponectin, soluble intracellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL-6), and accelerometer-determined sedentary time and moderate-vigorous physical activity (MVPA) were measured at baseline and after six-months. Linear regression analysis was used to investigate the independent cross-sectional and longitudinal associations of sedentary time with markers of inflammation. At baseline, associations between sedentary time and IL-6 were observed in men and women, an association that was attenuated following adjustment for waist circumference. After 6 months of follow-up, sedentary time was reduced by 0.4 ± 1.2 h per day in women, with the change in sedentary time predicting CRP at follow-up. Every hour decrease in sedentary time between baseline and six-months was associated with 24% (1, 48) lower CRP. No changes in sedentary time between baseline and 6 months were seen in men. CONCLUSIONS: Higher sedentary time is associated with IL-6 in men and women with type 2 diabetes, and reducing sedentary time is associated with improved levels of CRP in women. Interventions to reduce sedentary time may help to reduce inflammation in women with type 2 diabetes.

Distinct frontal systems for response inhibition, attentional capture, and error processing.

Stopping an action in response to an unexpected event requires both that the event is attended to, and that the action is inhibited. Previous neuroimaging investigations of stopping have failed to adequately separate these cognitive elements. Here we used a version of the widely used Stop Signal Task that controls for the attentional capture of stop signals. This allowed us to fractionate the contributions of frontal regions, including the right inferior frontal gyrus and medial frontal cortex, to attentional capture, response inhibition, and error processing. A ventral attentional system, including the right inferior frontal gyrus, has been shown to respond to unexpected stimuli. In line with this evidence, we reasoned that lateral frontal regions support attentional capture, whereas medial frontal regions, including the presupplementary motor area (pre-SMA), actually inhibit the ongoing action. We tested this hypothesis by contrasting the brain networks associated with the presentation of unexpected stimuli against those associated with outright stopping. Functional MRI images were obtained in 26 healthy volunteers. Successful stopping was associated with activation of the right inferior frontal gyrus, as well as the pre-SMA. However, only activation of the pre-SMA differentiated stopping from a high-level baseline that controlled for attentional capture. As expected, unsuccessful attempts at stopping activated the anterior cingulate cortex. In keeping with work in nonhuman primates these findings demonstrate that successful motor inhibition is specifically associated with pre-SMA activation.

Sedentary time, breaks in sedentary time and metabolic variables in people with newly diagnosed type 2 diabetes.

AIMS/HYPOTHESIS: We investigated whether objectively measured sedentary time and interruptions in sedentary time are associated with metabolic factors in people with type 2 diabetes. METHODS: We studied 528 adults (30-80 years) with newly diagnosed type 2 diabetes, who were participants in a diet and physical activity intervention. Waist circumference (WC), fasting HDL-cholesterol, insulin and glucose levels, HOMA of insulin resistance (HOMA-IR) and physical activity (accelerometer) were measured at baseline and at 6 months follow-up. Linear regression models were used to investigate cross-sectional and longitudinal associations of accelerometer-derived sedentary time and breaks in sedentary time (BST) with metabolic variables. RESULTS: In cross-sectional analyses each hour of sedentary time was associated with larger WC (unstandardised regression coefficient [B] [95% CI] 1.89 cm [0.94, 2.83]; p < 0.001), higher insulin (B = 8.22 pmol/l [2.80, 13.65]; p = 0.003) and HOMA-IR (B = 0.42 [0.14, 0.70]; p = 0.004), and lower HDL-cholesterol (B = -0.04 mmol/l [-0.06, -0.01]; p = 0.005). Adjustment for WC attenuated all associations. Each BST was associated with lower WC (B = -0.15 cm [- 0.24, -0.05]; p = 0.003) and there was evidence of a weak linear association with HDL-cholesterol, but no association with insulin levels or HOMA-IR. Volume of sedentary time at baseline predicted HDL-cholesterol (B = -0.05 mmol/l [-0.08, -0.01]; p = 0.007), insulin levels (B = 8.14 pmol/l [0.1.51, 14.78]; p = 0.016) and HOMA-IR (B = 0.49 [0.08, 0.90]; p = 0.020) at 6 months, though not WC. Baseline BST did not substantially predict any metabolic variables at follow-up. No change was seen in sedentary time or BST between baseline and 6 months follow-up. CONCLUSIONS/INTERPRETATION: Higher sedentary time is associated with a poorer metabolic profile in people with type 2 diabetes.

Diet or diet plus physical activity versus usual care in patients with newly diagnosed type 2 diabetes: the Early ACTID randomised controlled trial.

BACKGROUND: Lifestyle changes soon after diagnosis might improve outcomes in patients with type 2 diabetes mellitus, but no large trials have compared interventions. We investigated the effects of diet and physical activity on blood pressure and glucose concentrations. METHODS: We did a randomised, controlled trial in southwest England in adults aged 30-80 years in whom type 2 diabetes had been diagnosed 5-8 months previously. Participants were assigned usual care (initial dietary consultation and follow-up every 6 months; control group), an intensive diet intervention (dietary consultation every 3 months with monthly nurse support), or the latter plus a pedometer-based activity programme, in a 2:5:5 ratio. The primary endpoint was improvement in glycated haemoglobin A(1c)(HbA(1c)) concentration and blood pressure at 6 months. Analysis was done by intention to treat. This study is registered, number ISRCTN92162869. FINDINGS: Of 593 eligible individuals, 99 were assigned usual care, 248 the diet regimen, and 246 diet plus activity. Outcome data were available for 587 (99%) and 579 (98%) participants at 6 and 12 months, respectively. At 6 months, glycaemic control had worsened in the control group (mean baseline HbA(1c) percentage 6·72, SD 1·02, and at 6 months 6·86, 1·02) but improved in the diet group (baseline-adjusted difference in percentage of HbA(1c) -0·28%, 95% CI -0·46 to -0·10; p=0·005) and diet plus activity group (-0·33%, -0·51 to -0·14; p<0·001). These differences persisted to 12 months, despite less use of diabetes drugs. Improvements were also seen in bodyweight and insulin resistance between the intervention and control groups. Blood pressure was similar in all groups. INTERPRETATION: An intensive diet intervention soon after diagnosis can improve glycaemic control. The addition of an activity intervention conferred no additional benefit. FUNDING: Diabetes UK and the UK Department of Health.

Implications for psychedelic-assisted psychotherapy: functional magnetic resonance imaging study with psilocybin.

BACKGROUND: Psilocybin is a classic psychedelic drug that has a history of use in psychotherapy. One of the rationales for its use was that it aids emotional insight by lowering psychological defences. AIMS: To test the hypothesis that psilocybin facilitates access to personal memories and emotions by comparing subjective and neural responses to positive autobiographical memories under psilocybin and placebo. METHOD: Ten healthy participants received two functional magnetic resonance imaging scans (2 mg intravenous psilocybin v. intravenous saline), separated by approximately 7 days, during which they viewed two different sets of 15 positive autobiographical memory cues. Participants viewed each cue for 6 s and then closed their eyes for 16 s and imagined re-experiencing the event. Activations during this recollection period were compared with an equivalent period of eyes-closed rest. We split the recollection period into an early phase (first 8 s) and a late phase (last 8 s) for analysis. RESULTS: Robust activations to the memories were seen in limbic and striatal regions in the early phase and the medial prefrontal cortex in the late phase in both conditions (P<0.001, whole brain cluster correction), but there were additional visual and other sensory cortical activations in the late phase under psilocybin that were absent under placebo. Ratings of memory vividness and visual imagery were significantly higher after psilocybin (P<0.05) and there was a significant positive correlation between vividness and subjective well-being at follow-up (P<0.01). CONCLUSIONS: Evidence that psilocybin enhances autobiographical recollection implies that it may be useful in psychotherapy either as a tool to facilitate the recall of salient memories or to reverse negative cognitive biases.

Cytoplasmic dynein is required for poleward chromosome movement during mitosis in Drosophila embryos.

The movement of chromosomes during mitosis occurs on a bipolar, microtubule-based protein machine, the mitotic spindle. It has long been proposed that poleward chromosome movements that occur during prometaphase and anaphase A are driven by the microtubule motor cytoplasmic dynein, which binds to kinetochores and transports them toward the minus ends of spindle microtubules. Here we evaluate this hypothesis using time-lapse confocal microscopy to visualize, in real time, kinetochore and chromatid movements in living Drosophila embryos in the presence and absence of specific inhibitors of cytoplasmic dynein. Our results show that dynein inhibitors disrupt the alignment of kinetochores on the metaphase spindle equator and also interfere with kinetochore- and chromatid-to-pole movements during anaphase A. Thus, dynein is essential for poleward chromosome motility throughout mitosis in Drosophila embryos.

Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.

The positioning of centrosomes, or microtubule-organizing centres, within cells plays a critical part in animal development. Here we show that, in Drosophila embryos undergoing mitosis, the positioning of centrosomes within bipolar spindles and between daughter nuclei is determined by a balance of opposing forces generated by a bipolar kinesin motor, KLP61F, that is directed to microtubule plus ends, and a carboxy-terminal kinesin motor, Ncd, that is directed towards microtubule minus ends. This activity maintains the spacing between separated centrosomes during prometaphase and metaphase, and repositions centrosomes and daughter nuclei during late anaphase and telophase. Surprisingly, we do not observe a function for KLP61F in the initial separation of centrosomes during prophase. Our data indicate that KLP61F and Ncd may function by crosslinking and sliding antiparallel spindle microtubules in relation to one another, allowing KLP61F to push centrosomes apart and Ncd to pull them together.

Player position in American football influences the magnitude of mechanical strains produced in the location of chronic traumatic encephalopathy pathology: A computational modelling study.

American football players are frequently exposed to head impacts, which can cause concussions and may lead to neurodegenerative diseases such as chronic traumatic encephalopathy (CTE). Player position appears to influence the risk of concussion but there is limited work on its effect on the risk of CTE. Computational modelling has shown that large brain deformations during head impacts co-localise with CTE pathology in sulci. Here we test whether player position has an effect on brain deformation within the sulci, a possible biomechanical trigger for CTE. We physically reconstructed 148 head impact events from video footage of American Football games. Players were separated into 3 different position profiles based on the magnitude and frequency of impacts. A detailed finite element model of TBI was then used to predict Green-Lagrange strain and strain rate across the brain and in sulci. Using a one-way ANOVA, we found that in positions where players were exposed to large magnitude and low frequency impacts (e.g. defensive back and wide receiver), strain and strain rate across the brain and in sulci were highest. We also found that rotational head motion is a key determinant in producing large strains and strain rates in the sulci. Our results suggest that player position has a significant effect on impact kinematics, influencing the magnitude of deformations within sulci, which spatially corresponds to where CTE pathology is observed. This work can inform future studies investigating different player-position risks for concussion and CTE and guide design of prevention systems.

Pathways to the diagnosis of ovarian cancer in the UK: a cohort study in primary care.

OBJECTIVE: To identify the routes patients with ovarian cancer take between first symptom presentation and diagnosis. DESIGN: Cohort study. SETTING: The study took place in 39 general practices in Devon, UK. POPULATION: All ovarian cancer patients identified in the practices, with a diagnosis between 2000 and 2007 inclusive. METHODS: All patients had their cancer symptoms, referrals, and diagnoses identified and dated using their doctors' records. MAIN OUTCOME MEASURES: Numbers of patients taking specific routes to diagnosis, together with the time taken to diagnosis. RESULTS: Three main routes to diagnosis emerged. The first was the expected route of outpatient referral: 195 (92% of the total) had at least one of the seven ovarian cancer symptoms or an abdominal mass. A total of 123 (58%) were referred to a specialist, although only 65 (31%) were referred to a gynaecologist. Thirty-five (17%) were initially investigated within primary care by ultrasound scanning, and a further 35 (17%) were admitted as emergencies. The interval from first symptom to referral was similar across the different pathways, with a median (interquartile range) time between the first symptom presenting to primary care and first investigation or referral being 2.5 (0, 27.5) days. The median interval from first symptom reported in primary care to diagnosis was 74.5 (32, 159) days. CONCLUSIONS: Only a minority of ovarian cancer patients follow the expected route to diagnosis, of urgent referral to a gynaecologist. In most women, GPs rapidly identified the need to investigate. Avoidable delays generally occurred after the decision to investigate was made.

SARS-CoV-2 infection, clinical features and outcome of COVID-19 in United Kingdom nursing homes.

OBJECTIVES: To understand SARS-Co-V-2 infection and transmission in UK nursing homes in order to develop preventive strategies for protecting the frail elderly residents. METHODS: An outbreak investigation involving 394 residents and 70 staff, was carried out in 4 nursing homes affected by COVID-19 outbreaks in central London. Two point-prevalence surveys were performed one week apart where residents underwent SARS-CoV-2 testing and had relevant symptoms documented. Asymptomatic staff from three of the four homes were also offered SARS-CoV-2 testing. RESULTS: Overall, 26% (95% CI 22-31) of residents died over the two-month period. All-cause mortality increased by 203% (95% CI 70-336) compared with previous years. Systematic testing identified 40% (95% CI 35-46) of residents as positive for SARS-CoV-2, and of these 43% (95% CI 34-52) were asymptomatic and 18% (95% CI 11-24) had only atypical symptoms; 4% (95% CI -1 to 9) of asymptomatic staff also tested positive. CONCLUSIONS: The SARS-CoV-2 outbreak in four UK nursing homes was associated with very high infection and mortality rates. Many residents developed either atypical or had no discernible symptoms. A number of asymptomatic staff members also tested positive, suggesting a role for regular screening of both residents and staff in mitigating future outbreaks.

Transport of dendritic microtubules establishes their nonuniform polarity orientation.

The immature processes that give rise to both axons and dendrites contain microtubules (MTs) that are uniformly oriented with their plus-ends distal to the cell body, and this pattern is preserved in the developing axon. In contrast, developing dendrites gradually acquire nonuniform MT polarity orientation due to the addition of a subpopulation of oppositely oriented MTs (Baas, P. W., M. M. Black, and G. A. Banker. 1989. J. Cell Biol. 109:3085-3094). In theory, these minus-end-distal MTs could be locally nucleated and assembled within the dendrite itself, or could be transported into the dendrite after their nucleation within the cell body. To distinguish between these possibilities, we exposed cultured hippocampal neurons to nanomolar levels of vinblastine after one of the immature processes had developed into the axon but before the others had become dendrites. At these levels, vinblastine acts as a kinetic stabilizer of MTs, inhibiting further assembly while not substantially depolymerizing existing MTs. This treatment did not abolish dendritic differentiation, which occurred in timely fashion over the next two to three days. The resulting dendrites were flatter and shorter than controls, but were identifiable by their ultrastructure, chemical composition, and thickened tapering morphology. The growth of these dendrites was accompanied by a diminution of MTs from the cell body, indicating a net transfer of MTs from one compartment into the other. During this time, minus-end-distal microtubules arose in the experimental dendrites, indicating that new MT assembly is not required for the acquisition of nonuniform MT polarity orientation in the dendrite. Minus-end-distal microtubules predominated in the more proximal region of experimental dendrites, indicating that most of the MTs at this stage of development are transported into the dendrite with their minus-ends leading. These observations indicate that transport of MTs from the cell body is an essential feature of dendritic development, and that this transport establishes the nonuniform polarity orientation of MTs in the dendrite.

Mitosis, microtubules, and the matrix.

The mechanical events of mitosis depend on the action of microtubules and mitotic motors, but whether these spindle components act alone or in concert with a spindle matrix is an important question.

Inhibition of a mitotic motor compromises the formation of dendrite-like processes from neuroblastoma cells.

Microtubules in the axon are uniformly oriented, while microtubules in the dendrite are nonuniformly oriented. We have proposed that these distinct microtubule polarity patterns may arise from a redistribution of molecular motor proteins previously used for mitosis of the developing neuroblast. To address this issue, we performed studies on neuroblastoma cells that undergo mitosis but also generate short processes during interphase. Some of these processes are similar to axons with regard to their morphology and microtubule polarity pattern, while others are similar to dendrites. Treatment with cAMP or retinoic acid inhibits cell division, with the former promoting the development of the axon-like processes and the latter promoting the development of the dendrite-like processes. During mitosis, the kinesin-related motor termed CHO1/MKLP1 is localized within the spindle midzone where it is thought to transport microtubules of opposite orientation relative to one another. During process formation, CHO1/ MKLP1 becomes concentrated within the dendrite-like processes but is excluded from the axon-like processes. The levels of CHO1/MKLP1 increase in the presence of retinoic acid but decrease in the presence of cAMP, consistent with a role for the protein in dendritic differentiation. Moreover, treatment of the cultures with antisense oligonucleotides to CHO1/MKLP1 compromises the formation of the dendrite-like processes. We speculate that a redistribution of CHO1/MKLP1 is required for the formation of dendrite-like processes, presumably by establishing their characteristic nonuniform microtubule polarity pattern.

Identification of a microtubule-associated motor protein essential for dendritic differentiation.

The quintessential feature of the dendritic microtubule array is its nonuniform pattern of polarity orientation. During the development of the dendrite, a population of plus end-distal microtubules first appears, and these microtubules are subsequently joined by a population of oppositely oriented microtubules. Studies from our laboratory indicate that the latter microtubules are intercalated within the microtubule array by their specific transport from the cell body of the neuron during a critical stage in development (Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol. 130:93- 104). In addition, we have established that the mitotic motor protein termed CHO1/MKLP1 has the appropriate properties to transport microtubules in this manner (Sharp, D.J., R. Kuriyama, and P.W. Baas. 1996. J. Neurosci. 16:4370-4375). In the present study we have sought to determine whether CHO1/MKLP1 continues to be expressed in terminally postmitotic neurons and whether it is required for the establishment of the dendritic microtubule array. In situ hybridization analyses reveal that CHO1/MKLP1 is expressed in postmitotic cultured rat sympathetic and hippocampal neurons. Immunofluorescence analyses indicate that the motor is absent from axons but is enriched in developing dendrites, where it appears as discrete patches associated with the microtubule array. Treatment of the neurons with antisense oligonucleotides to CHO1/MKLP1 suppresses dendritic differentiation, presumably by inhibiting the establishment of their nonuniform microtubule polarity pattern. We conclude that CHO1/MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite.

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division.

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP(180), in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP(180) concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP(180) is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.