Molecular interplay between HURP and Kif18A in mitotic spindle regulation.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro , we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determined the binding mode of HURP to microtubules using Cryo-EM. The structure reveals that one HURP motif spans laterally across ß-tubulin, while a second motif binds between adjacent protofilaments. HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observed that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in spindle length control.

The mechanism and energetics of the dynein priming stroke.

Dyneins are an AAA+ motor responsible for motility and force generation toward the minus end of microtubules. Dynein motility is powered by nucleotide-dependent transitions of its linker domain, which transitions between straight (post-powerstroke) and bent (pre-powerstroke) conformations. To understand the dynamics and energetics of the linker, we performed all-atom molecular dynamics simulations of human dynein-2 primed for its power stroke. Simulations revealed that the linker can adopt either a bent conformation or a semi-bent conformation, separated by a 5.7 kT energy barrier. The linker cannot switch back to its straight conformation in the pre-powerstroke state due to a steric clash with the AAA+ ring. Simulations also showed that an isolated linker has a free energy minimum near the semi-bent conformation in the absence of the AAA+ ring, indicating that the linker stores energy as it bends and releases this energy during the powerstroke.

Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1-stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

Dyneins.

Dyneins are a family of motor proteins that carry out motility and force generation functions towards the minus end of microtubule filaments. Cytoplasmic dynein (dynein-1) is responsible for transporting intracellular cargos in the retrograde direction in the cytoplasm, anchoring several organelles to the microtubule network, driving nuclear migration in developing neurons, and orienting the mitotic spindle in dividing cells. All other dyneins are localized to cilia. Similar to dynein-1, dynein-2 walks along microtubules and drives intraflagellar transport in the retrograde direction. Other ciliary dyneins are positioned between adjacent microtubule doublets of the axoneme and power ciliary beating by sliding microtubules relative to each other. In this primer, we first highlight the structure, mechanism, and regulation of dynein-1, which is the best-characterized member of the dynein motor family, and then describe the unique features and cellular roles of other dyneins. We also discuss accessory proteins that regulate the activation and motility of dynein motors in different cellular contexts.

Nde1 promotes Lis1-mediated activation of dynein.

Cytoplasmic dynein drives the motility and force generation functions towards the microtubule minus end. The assembly of dynein with dynactin and a cargo adaptor in an active transport complex is facilitated by Lis1 and Nde1/Ndel1. Recent studies proposed that Lis1 relieves dynein from its autoinhibited conformation, but the physiological function of Nde1/Ndel1 remains elusive. Here, we investigate how human Nde1 and Lis1 regulate the assembly and subsequent motility of mammalian dynein using in vitro reconstitution and single molecule imaging. We find that Nde1 recruits Lis1 to autoinhibited dynein and promotes Lis1-mediated assembly of dynein-dynactin adaptor complexes. Nde1 can compete with the α2 subunit of platelet activator factor acetylhydrolase 1B (PAF-AH1B) for the binding of Lis1, which suggests that Nde1 may disrupt PAF-AH1B recruitment of Lis1 as a noncatalytic subunit, thus promoting Lis1 binding to dynein. Before the initiation of motility, the association of dynactin with dynein triggers the dissociation of Nde1 from dynein by competing against Nde1 binding to the dynein intermediate chain. Our results provide a mechanistic explanation for how Nde1 and Lis1 synergistically activate the dynein transport machinery.

TRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport.

Mitochondrial transport along microtubules is mediated by Miro1 and TRAK adaptors that recruit kinesin-1 and dynein-dynactin. To understand how these opposing motors are regulated during mitochondrial transport, we reconstitute the bidirectional transport of Miro1/TRAK along microtubules in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin and enhances the motility of kinesin-1 activated by its cofactor MAP7. We find that TRAK adaptors that recruit both motors move towards kinesin-1's direction, whereas kinesin-1 is excluded from binding TRAK transported by dynein-dynactin, avoiding motor tug-of-war. We also test the predictions of the models that explain how mitochondrial transport stalls in regions with elevated Ca2+. Transport of Miro1/TRAK by kinesin-1 is not affected by Ca2+. Instead, we demonstrate that the microtubule docking protein syntaphilin induces resistive forces that stall kinesin-1 and dynein-driven motility. Our results suggest that mitochondrial transport stalls by Ca2+-mediated recruitment of syntaphilin to the mitochondrial membrane, not by disruption of the transport machinery.

Studying Dynein Mechanochemistry with an Optical Trap.

Molecular motors generate force and mechanical work to perform some of the most energy-demanding cellular processes, such as whole cell motility and cell division. These motors experience resistance from the viscoelastic environment of the surrounding cytoplasm, and opposing forces that can originate from other motors bound to cytoskeleton. Optical trapping is the most widely used method to measure the force-generating and force-response characteristics of motor proteins. Here we present the methodologies of three different optical trapping assays we use to measure how forces originating from external factors affect the microtubule-detachment rate and velocity of dynein. We also briefly discuss the remaining challenges and future directions of optical trapping studies of dyneins and other microtubule-based motors.

Structural and functional insight into regulation of kinesin-1 by microtubule-associated protein MAP7.

Microtubule (MT)-associated protein 7 (MAP7) is a required cofactor for kinesin-1-driven transport of intracellular cargoes. Using cryo-electron microscopy and single-molecule imaging, we investigated how MAP7 binds MTs and facilitates kinesin-1 motility. The MT-binding domain (MTBD) of MAP7 bound MTs as an extended α helix between the protofilament ridge and the site of lateral contact. Unexpectedly, the MTBD partially overlapped with the binding site of kinesin-1 and inhibited its motility. However, by tethering kinesin-1 to the MT, the projection domain of MAP7 prevented dissociation of the motor and facilitated its binding to available neighboring sites. The inhibitory effect of the MTBD dominated as MTs became saturated with MAP7. Our results reveal biphasic regulation of kinesin-1 by MAP7 in the context of their competitive binding to MTs.

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that drives interactions between N proteins in condensates, and deletion of this region disrupts phase separation. We also identified small molecules that alter the size and shape of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

Structure and Mechanics of Dynein Motors.

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.

A giant adrenal hemorrhagic pseudocyst mimicking a parapelvic renal cyst: A case report and review of the literature.

Adrenal pseudocysts are rare, nonfunctional, asymptomatic cystic masses that originate from the adrenal gland and are usually located in the suprarenal area. They are usually incidentally discovered during imaging, but diagnosis can be challenging because they are similar to benign and malignant cystic lesions of the adrenal gland and adjacent organs. We describe a giant, adrenal hemorrhagic pseudocyst that was atypically located, extending from the middle to the lower poles of the kidney, admixed with a renal cortical cyst.

Comparison of Short and Mid-Term Mortality and Morbidity in Patients With Severe Aortic Stenosis (Intermediate/High Risk) Who Underwent Transcatheter Aortic Valve Replacement and Surgical Aortic Valve Replacement.

Treatment protocols for severe aortic valve stenosis include surgical aortic valve replacement (SAVR), balloon valvuloplasty, transcatheter aortic valve replacement (TAVR), and medical  treatment. Because the success rates are getting higher with both SAVR and TAVR, making the right treatment decision is important. This study retrospectively shows the short- (1 month) and mid-term (6 months) mortality and morbidity rate differences between 2 groups of patients, who arrived to our hospital from January 2014 through October 2018. The first group consists of 54 patients who underwent mid-high risk SAVR operations at Istanbul University-Cerrahpasa, Institute of Cardiology, Department of Cardiovascular Surgery. The second group consists of 57 patients who underwent TAVR at the Cardiology Department. Preoperative evaluation showed that the mean age of the SAVR group (71.5 years) was higher than the TAVR group (80 years). Also, the history of previous cardiac valve replacement surgery significantly was higher in the SAVR group than the TAVR group (P = .028). There were no significant differences between the remaining preoperative tests and diagnostic procedures. Of the patients who underwent SAVR, 3.7% experienced postoperative cardiac arrhythmias, while the 17.5% of patients from the TAVR group experienced cardiac arrhythmias after the procedure. This difference between the groups were statistically significant. Mortality rate was 9.3% in the SAVR group and 5.3% in the TAVR group. The mortality rate was not statistically different between the groups. There was no significant difference between the groups in the means of neurological incidents. The TAVR group had more vascular complications (17.9% to none) and pacemaker implantations (21.4% to 1.9%). Minor or major bleeding was the most common reason for admission to the hospital after SAVR. Seven out of 10 patients experienced bleeding. Aortic regurgitation was more common in the TAVR group at the first and sixth month following the procedure. Ratios between the gradient values were higher in the SAVR group (P < .001). Peak gradient values at the sixth month following the procedure were lower than the values of the first month (P < .040). Aortic regurgitation symptoms increased with patients at the mid-term follow-up appointment. To prevent the vascular complications in the TAVR group, preoperative peripheral vascular examination thoroughly should be performed. Considering that bleeding disorders are the main reason the SAVR group arrived to the hospital, INR values should closely be monitored. There seems to be no mortality difference between the groups at the six-month follow up, but studies should continue with more patients and long-term results.

Lis1 activates dynein motility by modulating its pairing with dynactin.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favours the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.

Cargo adaptors regulate stepping and force generation of mammalian dynein-dynactin.

Cytoplasmic dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when dynein assembles with dynactin and a cargo adaptor. It was unclear how active dynein-dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that dynein-dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple dyneins collectively bear a large amount of tension because the backward stepping rate of dynein is insensitive to load. Recruitment of two dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail-tail interactions between two closely packed dyneins. Our results show that cargo adaptors modulate dynein motility and force generation for a wide range of cellular functions.

Kinesin and dynein use distinct mechanisms to bypass obstacles.

Kinesin-1 and cytoplasmic dynein are microtubule (MT) motors that transport intracellular cargoes. It remains unclear how these motors move along MTs densely coated with obstacles of various sizes in the cytoplasm. Here, we tested the ability of single and multiple motors to bypass synthetic obstacles on MTs in vitro. Contrary to previous reports, we found that single mammalian dynein is highly capable of bypassing obstacles. Single human kinesin-1 motors fail to avoid obstacles, consistent with their inability to take sideways steps on to neighboring MT protofilaments. Kinesins overcome this limitation when working in teams, bypassing obstacles as effectively as multiple dyneins. Cargos driven by multiple kinesins or dyneins are also capable of rotating around the MT to bypass large obstacles. These results suggest that multiplicity of motors is required not only for transporting cargos over long distances and generating higher forces, but also for maneuvering cargos on obstacle-coated MT surfaces.

Directionality of dynein is controlled by the angle and length of its stalk.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.

Correlation between Breast Arterial Calcification and the 10-year fatal cardiovascular risk by means of the SCORE Risk System.

OBJECTIVES: The aim of this study was to investigate the relationship between Breast Arterial Calcification (BAC) on mammography and the 10-year fatal Cardiovascular Disease (CVD) risk by using SCORE risk system. METHODS: The study was conducted from September 2013 to July 2014. A total of 66 women with BAC and 66 age-matched controls without BAC were analyzed. The groups were compared with respect to demographics, clinical, reproductive, laboratory parameters, and 10-year fatal CVD risk. RESULTS: The mean ages of the women in the study was 54.0 years (40-85 years). Hypertension, systolic blood pressure, levels of serum total cholesterol and the calculated SCORE risk were higher in the BAC (+) group than in the BAC (-) group (p=0.04, p=0.031, p=0.046, and p=0.038 respectively). Multivariate analysis showed that none of them was independent factor of BAC on mammograms, only the 10-year fatal CVD risk was close to being statistically significant (OR:1.17, CI:0.98-1.38, p=0.06). CONCLUSION: BAC on mammography was found to be related to the 10-year fatal CVD risk as calculated by the SCORE risk score system. Additional large-scale prospective studies are required to further assess whether BAC can be considered a useful screening tool for CVD risk prediction in women who screened for breast cancer by mammography.

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

Impact of continuation of metformin prior to elective coronary angiography on acute contrast nephropathy in patients with normal or mildly impaired renal functions.

OBJECTIVE: Discontinuation of metformin treatment in patients scheduled for elective coronary angiography (CAG) is controversial because of post-procedural risks including acute contrast-induced nephropathy (CIN) and lactic acidosis (LA). This study aims to discuss the safety of continuing metformin treatment in patients undergoing elective CAG with normal or mildly impaired renal functions. METHODS: Our study was designed as a single-centered, randomized, and observational study including 268 patients undergoing elective CAG with an estimated glomerular filtration rate of >60 mL/min/1.73 m2. Of these patients, 134 continued metformin treatment during angiography, whereas 134 discontinued it 24 h before the procedure. CIN was defined as either a 25% relative increase in serum creatinine levels from the baseline or a 0.5 mg/dL increase in the absolute value that measured 48 h after CAG. Logistic regression analysis was performed to identify independent predictors of CIN and LA after CAG. RESULTS: Both groups were comparable in terms of demographics and laboratory values. CIN at 48 h was 8% (11/134) in the metformin continued group and 6% (8/134) in the metformin discontinued group (p=0.265). Patients in neither of the groups developed metformin-induced LA. Based on multiple regression analysis, the ejection fraction [p=0.029, OR: 0.760; 95% CI (0.590-0.970)] and contrast volume [p=0.016, OR: 0.022 95% CI (0.010-0.490)] were independent predictors of CIN. CONCLUSION: Patients scheduled for elective CAG with normal or mildly impaired renal functions and preserved left ventricular ejection fraction (>40%) may safely continue metformin treatment.