Spindle Positioning Research Articles

THE ROLE OF THE POLARITY PROTEIN PAR3 IN HAEMATOPOIESIS AND LEUKAEMIA

Cell polarity is a critical feature of multiple aspects of cell biology e.g. cell division and differentiation. Disrupted polarity network is well established in epithelial cancers where the polarity regulators act as tumour-suppressors or oncogenes. In the haematopoietic system, polarity proteins regulate asymmetric cell division (ACD) of haematopoietic stem cells (HSCs). ACD is central to the maintenance of normal haematopoiesis, ensuring the retention of self-renewal and differentiation potentials of HSCs. Disrupted ACD is associated with leukaemia transformation as leukemic cells lose the ability to differentiate and instead gain the ability to self-renew. Considering this and the role of polarity proteins in ACD, understanding how they regulate HSCs ACD or contribute to leukaemia is of major importance. We examined the role of polarity protein PAR3 in acute myeloid leukaemia (AML). To determine the role of PAR3 loss in haematopoiesis, we utilised a Par3 conditional knockout mouse model. For the human model, Par3 was knocked out in AML cell lines using CRISPR/Cas9. We found that PAR3 is differentially expressed in haematopoietic progenitors and their more differentiated progeny. PAR3 loss in a pre-clinical model of murine AML led to a significant increase in the proportion and number of granulocyte/macrophage progenitors, which are the hyperproliferative cancer stem cells. This was associated with deregulation of MAPK and HIPPO signalling pathways in PAR3 KO animals. To study the cellular and molecular mechanisms of PAR3 in human AML, we have successfully knocked out the Par3 in several human AML cell lines. The role of PAR3 in ACD is being studied by examining the cell cycle status using live cell imaging, cell differentiation, polarised inheritance of cell fate determinants, and mitotic spindle positioning. In conclusion, PAR3 acts as a tumour suppressor in AML.

Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells

Proper spindle positioning is crucial for spatial cell division control. Spindle positioning in human cells relies on a ternary complex comprising Gαi1–3, LGN and NuMA, which anchors dynein at the cell cortex, thus enabling pulling forces to be exerted on astral microtubules. We develop a live imaging siRNA-based screen using stereotyped fibronectin micropatterns to uncover components modulating spindle positioning in human cells, testing 1280 genes, including all kinases and phosphatases. We thus discover 16 components whose inactivation dramatically perturbs spindle positioning, including tyrosine receptor kinase 3 (TYRO3) and cyclin G associated kinase (GAK). TYRO3 depletion results in excess NuMA and dynein at the cortex during metaphase, similar to the effect of blocking the TYRO3 downstream target phosphatidylinositol 3-kinase (PI3K). Furthermore, depletion of GAK leads to impaired astral microtubules, similar to the effect of downregulating the GAK-interactor Clathrin. Overall, our work uncovers components and mechanisms governing spindle positioning in human cells.

High-resolution imaging reveals how the spindle midzone impacts chromosome movement.

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

The DNA damage checkpoint and the spindle position checkpoint: guardians of meiotic commitment.

Exogenous signals induce cells to enter the specialized cell division process of meiosis, which produces haploid gametes from diploid progenitor cells. Once cells initiate the meiotic divisions, it is imperative that they complete meiosis. Inappropriate exit from meiosis and entrance into mitosis can create polyploid cells and can lead to germline tumors. Saccharomyces cerevisiae cells enter meiosis when starved of nutrients but can return to mitosis if provided nutrient-rich medium before a defined commitment point. Once past the meiotic commitment point in prometaphase I, cells stay committed to meiosis even in the presence of a mitosis-inducing signal. Recent research investigated the maintenance of meiotic commitment in budding yeast and found that two checkpoints that do not normally function in meiosis I, the DNA damage checkpoint and the spindle position checkpoint, have crucial functions in maintaining meiotic commitment. Here, we review these findings and discuss how the mitosis-inducing signal of nutrient-rich medium could activate these two checkpoints in meiosis to prevent inappropriate meiotic exit.

Fuzziy Digital Filter for Robotic Manipulator Operation

In this article it is described the operation principle of a robotic manipulator ARMino device and the connection diagram of its electrical components. The device includes: An Arduino Mega control board, four servos, four potentiometers, a prototyping board, a computer. Turning the shafts on the potentiometer adjusts the position of the servo spindles. When the voltage on the potentiometer’s pin changes, the voltage at the analog inputs of the microcontroller changes. Then, in the microcontroller, the voltage is scaled to the value of the servo rotation angle. After that, the joints of the robotic manipulator are rotated. During the operation of the ARMino robotiсarm, a contact bounce problem appeared, significantly reducing the accuracy of positioning and the smooth movement of the ARMino joints. To solve this problem, a digital filter was developed. This article describes the digital filter working algorithm, which consists of four steps. One of the steps consists on finding the digital filter coefficients, which regulate the signal voltage level transmitted to the servo motors, and its transition process time which forms the signal edge. The main problem developing a digital filter is that the standard procedure of finding the digital filter coefficients, the coefficients are given by a recommended range of values, which complicates choosing from this range, a single value and transmitting it to the servos. To solve this problem, a fuzzy digital filter was developed, the algorithm of which consists of six steps. The first step determines the input variables degree of truth. The second step is to calculate the degrees of truth of the fuzzy rules preconditions. The third step is to calculate the degrees of truth of the fuzzy rules conclusions by using the process of finding the maximum values. The fourth step is the defuzzification stage in which a precise value of the fuzzy digital filter coefficient is calculated. The fifth step is the output voltage transmitted to the servos. In the sixth step, the output voltage in the microcontroller is converted to the angle value and the servo is given the command to rotate. This article presents numerical simulation of the fuzzy digital filter algorithm, using as an example the servo responsible of the ARMino base rotation. Experimental studies on the functioning of the fuzzy digital filter have been carried out, confirming the expediency of its use. The graphics of the transition process of the robotic manipulator base movement without and with the use of a digital filter are given.

Loss of PKC mu function induces cytoskeletal defects in mouse oocyte meiosis.

Cytoskeleton which includes microtubule and actin filaments plays important roles during mammalian oocyte maturation. In the present study, we showed that protein kinase C mu (PKC mu) was one potential key molecule which affected cytoskeleton dynamics in mouse oocytes. Our results showed that PKC mu expressed and localized at the poles of the spindle during oocyte maturation, and PKC mu expression reduced in the oocytes from 6-month-old mice or 24 hr in vitro culture. We knocked down the expression of PKC mu in oocytes using morpholino injection to explore the relationship between PKC mu and subcellular structure defects. The loss of PKC mu reduced oocyte maturation competence, showing with decreased polar body extrusion rate and increased rate of symmetric division. Further analysis indicated that PKC mu decrease caused the spindle organization defects, and this could be confirmed by the decreased tubulin acetylation level. Moreover, we found that PKC mu affected the phosphorylation level of cofilin for actin assembly, which further affected cytoplasmic actin distribution and spindle positioning. In summary, our data indicated that PKC mu is one key factor for oocyte maturation through its roles on the spindle organization and actin filament distribution.

Mechanisms of Spindle Positioning: Lessons from Worms and Mammalian Cells.

Proper positioning of the mitotic spindle is fundamental for specifying the site for cleavage furrow, and thus regulates the appropriate sizes and accurate distribution of the cell fate determinants in the resulting daughter cells during development and in the stem cells. The past couple of years have witnessed tremendous work accomplished in the area of spindle positioning, and this has led to the emergence of a working model unravelling in-depth mechanistic insight of the underlying process orchestrating spindle positioning. It is evident now that the correct positioning of the mitotic spindle is not only guided by the chemical cues (protein–protein interactions) but also influenced by the physical nature of the cellular environment. In metazoans, the key players that regulate proper spindle positioning are the actin-rich cell cortex and associated proteins, the ternary complex (Gα/GPR-1/2/LIN-5 in Caenorhabditis elegans, Gαi/Pins/Mud in Drosophila and Gαi1-3/LGN/NuMA in humans), minus-end-directed motor protein dynein and the cortical machinery containing myosin. In this review, I will mainly discuss how the abovementioned components precisely and spatiotemporally regulate spindle positioning by sensing the physicochemical environment for execution of flawless mitosis.

Mechanisms of Astral Microtubule Regulation by Kinesin Motor Proteins

During mitotic division cells construct a bipolar spindle that aligns and accurately divides the chromosomes. Correct construction and positioning of the spindle is important to prevent segregation errors. Astral microtubules connect the spindle poles to the cell cortex and provide the pushing and pulling forces required to accurately position the spindle in the cell. Two kinesin motor proteins, Kif18b (Kinesin-8) and MCAK (Kinesin-13), are important regulators of astral microtubule length and spindle positioning in human cells. We are investigating the mechanism by which Kif18b regulates microtubule length both in vitro and in cells. We have found that Kif18b is a highly processive motor that targets microtubule plus ends and alters their dynamic behaviour. Kif18b is regulated through its C-terminal tail region and this is important for targeting to astral microtubule plus tips. Although Kif18b motor domains are capable of depolymerising stable microtubules its tail domain counteracts this ability. We present new data exploring the interaction between Kif18b and microtubule plus tip proteins and propose a model for Kif18b mediated astral microtubule regulation.

The DNA Damage Checkpoint and the Spindle Position Checkpoint Maintain Meiotic Commitment in Saccharomyces cerevisiae

During meiosis, diploid progenitor cells undergo one round of DNA replication followed by two rounds of chromosome segregation to form haploid gametes. Once cells initiate the meiotic divisions, it is imperative that they finish meiosis. A failure to maintain meiosis can result in highly aberrant polyploid cells, which could lead to oncogenesis in the germline. How cells stay committed to finishing meiosis, even in the presence of a mitosis-inducing signal, is poorly understood. We addressed this question in budding yeast, in which cells enter meiosis when starved. Ifnutrient-rich medium is added before a defined commitment point in mid-prometaphase I, they can return to mitosis. Cells in stages beyond the commitment point will finish meiosis, even with nutrient addition. Because checkpoints are signaling pathways known to couple cell-cycle processes with one another, we asked if checkpoints could ensure meiotic commitment. We find that two checkpoints with well-defined functions in mitosis, the DNA damage checkpoint and the spindle position checkpoint, have crucial roles in meiotic commitment. With nutrient-rich medium addition at stages beyond the commitment point, cells that are deficient in both checkpoints because they lack Rad53 and either Bub2, Bfa1, or Kin4 can return to mitotic growth and go on to form polyploid cells. The results demonstrate that the two checkpoints prevent cells from exiting meiosis in the presence of a mitosis-inducing signal. This study reveals a previously unknown function for the DNA damage checkpoint and the spindle position checkpoint in maintaining meiotic commitment.

Development of Real Time Machine Tools Component Utilization Data Acquisition for developing Dynamic Model of Maintenance Scheduling

Maintenance scheduling accuracy of CNC machine tools components should be calculated based on actual data utilization of each component. Actual usage of each component can be approached by component grouping based on machine operation phase utilization, which is divided into Power-On, cutting and noncutting phase. This paper describes a study on development of machine monitoring data utilization for obtaining real time information of spindle and machine axis positions as well as current consumption of spindle servo motor. Data collection was conducted by a termination method on wiring feedback control of spindle encoder signal and axes encoder signals. On the other hand, current consumption of spindle servo motor was measured by a CT current transducer. Afterward, a WEMOS microcontroller was used to process and to transfer data wirelessly to a Raspberry which acted as a broker. The data will used to update the data status of CNC Machine Tools utilization database, where it was communicated by using a MQTT protocol. A monitoring system has been developed and resulted the real time information of machine phase utilization. It will be further utilized as primary data input for building a dynamic maintenance model.

A neurodevelopmental TUBB2B β-tubulin mutation impairs Bim1 (yeast EB1)-dependent spindle positioning.

ABSTRACTMalformations of the human cerebral cortex can be caused by mutations in tubulins that associate to compose microtubules. Cerebral cortical folding relies on neuronal migration and on progenitor proliferation partly dictated by microtubule-dependent mitotic spindle positioning. A single amino acid change, F265L, in the conserved TUBB2B β-tubulin gene has been identified in patients with abnormal cortex formation. A caveat for studying this mutation in mammalian cells is that nine genes encode β-tubulin in human. Here, we generate a yeast strain expressing F265L tubulin mutant as the sole source of β-tubulin. The F265L mutation does not preclude expression of a stable β-tubulin protein which is incorporated into microtubules. However, impaired cell growth was observed at high temperatures along with altered microtubule dynamics and stability. In addition, F265L mutation produces a highly specific mitotic spindle positioning defect related to Bim1 (yeast EB1) dysfunction. Indeed, F265L cells display an abnormal Bim1 recruitment profile at microtubule plus-ends. These results indicate that the F265L β-tubulin mutation affects microtubule plus-end complexes known to be important for microtubule dynamics and for microtubule function during mitotic spindle positioning.

Numerical assessment of ejector performance enhancement by means of combined adjustable-geometry and bypass methods

The relatively low entrainment ratio is the key problem that confines the ejector development in the industry. In this study, the adjustable-geometry ejectors (with three different spindle positions) and ejectors with bypass (with two different bypass inlet installations) were adopted to promote the entrainment ratio of an ejector. The performance of the adjustable-geometry combined with bypass was also investigated. Results showed that the adjustable-geometry ejector and ejector with bypass could improve the entrainment ratio, and both ejectors reached a peak value. Compared with the original ejector, the maximum increment was 51.47% for the adjustable-geometry ejector and 26.0% for the ejector with bypass, respectively. Meanwhile, the combined ejector increased the entrainment ratio, although the combination was only effective at higher induced pressures than that of the adjustable-geometry ejector without bypass. The maximum increment was up to 74.5% compared to that of the original ejector.

Numerical study on spindle positioning using phase field method

A method of numerical simulation of cell division using phase fields is presented. The cell division plane is obtained as a result of the spindle position and orientation considered with the spatial distribution of the activated cortical force generators and the dividing cell shape. To exemplify the application of the proposed method, numerical simulations of the development of cysts and early embryos are performed. The numerical results demonstrate that the activated cortical force generators that are localized at the lateral cortices of the epithelial cells lead to the formation of a single central lumen. It is additionally shown that the linear distribution of the activated cortical force generators along the animal-vegetal axis of a spherical cell engenders a similar cell proliferation of the divided embryo generated by the 32 cell period in a sea cucumber.

Budding Yeast BFA1 Has Multiple Positive Roles in Directing Late Mitotic Events.

The proper regulation of cell cycle transitions is paramount to the maintenance of cellular genome integrity. In Saccharomyces cerevisiae, the mitotic exit network (MEN) is a Ras-like signaling cascade that effects the transition from M phase to G1 during the cell division cycle in budding yeast. MEN activation is tightly regulated. It occurs during anaphase and is coupled to mitotic spindle position by the spindle position checkpoint (SPoC). Bfa1 is a key component of the SPoC and functions as part of a two-component GAP complex along with Bub2. The GAP activity of Bfa1-Bub2 keeps the MEN GTPase Tem1 inactive in cells with mispositioned spindles, thereby preventing inappropriate mitotic exit and preserving genome integrity. Interestingly, a GAP-independent role for Bfa1 in mitotic exit regulation has been previously identified. However the nature of this Bub2-independent role and its biological significance are not understood. Here we show that Bfa1 also activates the MEN by promoting the localization of Tem1 primarily to the daughter spindle pole body (dSPB). We demonstrate that the overexpression of BFA1 is lethal due to defects in Tem1 localization, which is required for its activity. In addition, our studies demonstrate a Tem1-independent role for Bfa1 in promoting proper cytokinesis. Cells lacking TEM1, in which the essential mitotic exit function is bypassed, exhibit cytokinesis defects. These defects are suppressed by the overexpression of BFA1. We conclude that Bfa1 functions to both inhibit and activate late mitotic events.

Improvement of technology of manufacturing and control of quality of mirrors-reflectors from aluminium alloy

At present, the technology of diamond blade whetting with nano-sized roughness is widely used at the manufaсturing of metal-optical products, first of all, mirror-reflectors for “transportation” of powerful laser energy flows. Optimum material for mirror-reflectors is an aluminum alloy AMg2, which surface purity, is affected by the quality of preliminary mechanical heat treatment during superfinishing treatment by diamond whetting.Preliminary machining of the surface with a carbide cutter and finishing with a diamond cutter (with a radius of curvature of the blade less than 0.05 μm) were performed on a precision lathe of the MK 6501 model with a vertical spindle position on an air bearing. Thermal treatment was carried out in the laboratory electric furnace SNOL 58/350. Various modes of preliminary heat treatment, machining with a carbide cutter and finishing with a diamond cutter of substrates (20×20×7 mm3) were tested. The surface state analysis was carried out using the PMT-3 microhardness tester, the SolverPro P47 atomic-force microscope (AFM), and the experimental probe-electrometry device. The control of the electrophysical parameters of the surface was carried out by recording the distribution of the electron work function (RWF) by the contact potential difference with the processing by the microprocessor measuring transducer of electrostatic potentials. The recorded changes in the RWF characterize the physic-chemical and mechanical parameters of the surface of mirrors and indicate the presence of a different type and nature of defects.Modified preliminary mechanical-thermal treatment allowed to improve the cleanliness of surface treatment of substrates. Finishing nanoscale diamond blade processing, including the complete removal of the surface layer that was disturbed by previous operations, bring to the greatest possible improvement in the quality of the surface in terms of the uniformity of the distribution of its electrophysical properties. As a result, according to the values and changes of the RWF, achievement of the specified performance characteristics of the product surface was monitored in order to optimize the technological processing modes in accordance with the functional designations of the devices.The methods for increasing the efficiency of nanoscale diamond blade processing and performing researches of the electrophysical properties of the surface to control defects in the manufacture of metal reflector mirrors with high reflectivity and radiation strength for operation under extreme conditions.

Microtubule Feedback and LET-99-Dependent Control of Pulling Forces Ensure Robust Spindle Position

During asymmetric division of the Caenorhabditis elegans zygote, to properly distribute cell fate determinants, the mitotic spindle is asymmetrically localized by a combination of centering and cortical-pulling microtubule-mediated forces, the dynamics of the latter being regulated by mitotic progression. Here, we show a, to our knowledge, novel and additional regulation of these forces by spindle position itself. For that, we observed the onset of transverse spindle oscillations, which reflects the burst of anaphase pulling forces. After delaying anaphase onset, we found that the position at which the spindle starts to oscillate was unchanged compared to control embryos and uncorrelated to anaphase onset. In mapping the cortical microtubule dynamics, we measured a steep increase in microtubule contact density after the posterior centrosome reached the critical position of 70% of embryo length, strongly suggesting the presence of a positional switch for spindle oscillations. Expanding a previous model based on a force-generator temporal control, we implemented this positional switch and observed that the large increase in microtubule density accounted for the pulling force burst. Thus, we propose that the spindle position influences the cortical availability of microtubules on which the active force generators, controlled by cell cycle progression, can pull. Importantly, we found that this positional control relies on the polarity-dependent LET-99 cortical band, the boundary of which could be probed by microtubules. This dual positional and temporal control well accounted for our observation that the oscillation onset position resists changes in cellular geometry and moderate variations in the active force generator number. Finally, our model suggests that spindle position at mitosis end is more sensitive to the polarity factor LET-99, which restricts the region of active force generators to a posterior-most region, than to microtubule number or force generator number/activity. Overall, we show that robustness in spindle positioning originates in cell mechanics rather than biochemical networks.

The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere of C. elegans

The PAR-1 kinase of C. elegans is localized to the posterior of the one-cell embryo and its mutations affect asymmetric spindle placement and partitioning of cytoplasmic components in the first cell cycle. However, par-1 mutations do not cause failure to restrict the anterior PAR polarity complex to the same extent as mutations in the posteriorly localized PAR-2 protein. Further, it has been difficult to examine the role of PAR-1 in subsequent divisions due to the early defects in par-1 mutant embryos. Here we show that the PIG-1 kinase acts redundantly with PAR-1 to restrict the anterior PAR-3 protein for normal polarity in the one-cell embryo. By using a temperature sensitive allele of par-1, which exhibits enhanced lethality when combined with a pig-1 mutation, we have further explored roles for these genes in subsequent divisions. We find that both PIG-1 and PAR-1 regulate spindle orientation in the EMS blastomere of the four-cell stage embryo to ensure that it undergoes an asymmetric division. In this cell, PIG-1 and PAR-1 act in parallel pathways for spindle positioning, PIG-1 in the MES-1/SRC-1 pathway and PAR-1 in the Wnt pathway.

Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Meiotic spindle position and second polar body orientation enable the prediction of embryonic developmental potential and embryo euploidy

Optogenetic dissection of mitotic spindle positioning in vivo.

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Shining light on spindle positioning.

Optogenetic approaches are leading to a better understanding of the forces that determine the plane of cell division.