Kinesin Function Research Articles

The Application of Kinesin Inhibitors in Medical Issues.

Kinesins are a group of motor proteins in charge of several crucial functions in the cell. These proteins often bind to microtubules and perform their functions using the energy produced by ATP hydrolysis. One function of mitotic kinesin, a subclass of kinesin that is expressed during cell division at the mitotic phase, is to create the mitotic spindle. Uncontrolled cell growth is one trait of cancerous cells. Traditional anticancer medications still used in clinics include taxanes (paclitaxel) and vinca alkaloids (vincristine, vinblastine), which interfere with microtubule dynamics. However, because non-dividing cells like post-mitotic neurons contain microtubules, unwanted side effects like peripheral neuropathy are frequently found in patients taking these medications. More than ten members of the mitotic kinesin family play distinct or complementary roles during mitosis. The mitotic kinesin family's KSP, or Eg5, is regarded as its most dramatic target protein. The current work systematically reviews the use of kinesin inhibitors in the medical field. The challenges of KSP and the practical solutions are also examined, and the outcomes of the previous works are reported. The significant gaps and shortcomings of the related works are also highlighted, which can be an onset topic for future works.

Involvement of kinesins in skeletal dysplasia: a review.

Skeletal dysplasias are group of rare genetic diseases resulting from mutations in genes encoding structural proteins of the cartilage extracellular matrix (ECM), signaling molecules, transcription factors, epigenetic modifiers, and several intracellular proteins. Cell division, organelle maintenance, and intracellular transport are all orchestrated by the cytoskeleton-associated proteins, and intracellular processes affected through microtubule-associated movement are important for the function of skeletal cells. Among microtubule-associated motor proteins, kinesins in particular have been shown to play a key role in cell cycle dynamics, including chromosome segregation, mitotic spindle formation, and ciliogenesis, in addition to cargo trafficking, receptor recycling, and endocytosis. Recent studies highlight the fundamental role of kinesins in embryonic development and morphogenesis and have shown that mutations in kinesin genes lead to several skeletal dysplasias. However, many questions concerning the specific functions of kinesins and their adaptor molecules as well as specific molecular mechanisms in which the kinesin proteins are involved during skeletal development remain unanswered. Here we present a review of the skeletal dysplasias resulting from defects in kinesins and discuss the involvement of kinesin proteins in the molecular mechanisms that are active during skeletal development.

Regulatory mechanisms of kinesin function at varying pH

Regulatory mechanisms of kinesin function at varying pH

Cytokinesis in Trypanosoma brucei relies on an orphan kinesin that dynamically crosslinks microtubules

Many single-celled eukaryotes have complex cell morphologies defined by microtubules arranged into higher-order structures. The auger-like shape of the parasitic protist Trypanosoma brucei (T.brucei) is mediated by a parallel array of microtubules that underlies the plasma membrane. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the ingressing cleavage furrow and is essential for the completion of cytokinesis. We have characterized the biophysical properties of a truncated KLIF construct invitro to gain mechanistic insight into the function of this novel kinesin. We find that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked by KLIF in an antiparallel orientation are translocated relative to one another, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we find that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new cell posterior, which is an essential requirement for the completion of cytokinesis in T.brucei.

Electro-detachment of kinesin motor domain from microtubule in silico

Kinesin is a motor protein essential in cellular functions, such as intracellular transport and cell-division, as well as for enabling nanoscopic transport in bio-nanotechnology. Therefore, for effective control of function for nanotechnological applications, it is important to be able to modify the function of kinesin. To circumvent the limitations of chemical modifications, here we identify another potential approach for kinesin control: the use of electric forces. Using full-atom molecular dynamics simulations (247,358 atoms, total time ∼ 4.4 μs), we demonstrate, for the first time, that the kinesin-1 motor domain can be detached from a microtubule by an intense electric field within the nanosecond timescale. We show that this effect is field-direction dependent and field-strength dependent. A detailed analysis of the electric forces and the work carried out by electric field acting on the microtubule–kinesin system shows that it is the combined action of the electric field pulling on the β-tubulin C-terminus and the electric-field-induced torque on the kinesin dipole moment that causes kinesin detachment from the microtubule. It is shown, for the first time in a mechanistic manner, that an electric field can dramatically affect molecular interactions in a heterologous functional protein assembly. Our results contribute to understanding of electromagnetic field–biomatter interactions on a molecular level, with potential biomedical and bio-nanotechnological applications for harnessing control of protein nanomotors.

OsTUB1 confers salt insensitivity by interacting with Kinesin13A to stabilize microtubules and ion transporters in rice.

Salt stress is one of the major environmental factors limiting plant growth and development. Although microtubule (MT) organization is known to be involved in response to salt stress, few tubulin genes have been identified that confer salt insensitivity in plants. In this study, we identified a MT encoding gene, OsTUB1, that increased the survival rate of rice plants under salt stress by stabilizing MT organization and ion transporters. We found that OsTUB1 interacted with Kinesin13A protein, which was essential for OsTUB1-regulated MT organization under salt stress. Further molecular evidence revealed that a OsTUB1-Kinesin13A complex protected rice from salt stress by sustaining membrane-localized Na+ transporter OsHKT1;5, a key regulator of ionic homeostasis. Our results shed light on the function of tubulin and kinesin in regulating MT organization and stabilizing Na+ transporters and Na+ flux at the plasma membrane in rice. The identification of the OsTUB1-Kinesin13A complex provides novel genes for salt insensitivity rice breeding in areas with high soil salinity.

Abstract 2419: Understanding the role of DCLK1 mediated invadopodia regulation in HNSCC

Abstract Despite therapeutic advances, head and neck squamous cell carcinoma (HNSCC) is marked by high mortality, particularly with increasing stage. Locoregional invasion, an early step in metastasis, is driven by the development of finger-like processes on the tumor cell known as invadopodia. Mature, active invadopodia secrete matrix metalloproteases (MMP) that enable extension of the cell into the extracellular matrix (ECM). Kinesins haul RAB-bound vesicles containing MMPs along microtubules the length of invadopodia for secretion to the most distal aspect of the invadopodia. It is unclear how these kinesins are modulated within invadopodia. In preliminary studies on patient samples, we identified high levels of doublecortin like kinase (DCLK1) at the invasive front of HNSCC. We hypothesize DCLK1 supports the function of kinesins as a mechanism of invasion. DCKL1 was knocked down in HNSCC lines with a short hairpin RNA (shDCLK1). Invasion and migration assays were performed in shDCLK1 and shcontrol tumor cells by utilizing a transwell assay. Confocal microscopy using Leica LAS X, was performed to assess colocalization of DCLK1 and markers of mature invadopodia (cortactin, TKS5, MT1-MMP) in HNSCC lines for. . To validate the association of DCLK1 with Kif16B and RAB40b, we carried out a co-immunoprecipitation analysis. Finally, conditioned media from shDCLK1 and shControl HNSCC cells was used to assess MMP activity using gelatin zymography and MMP profiler arrays. Transwell assay demonstrates reduced movement in shDCLK1 compared to shcontrol tumor cells (p <0.0001 invasion, p <0.001 migration). Confocal microscopy shows colocalization of MMP 9 and DCLK1 to invadopodia. DCLK1 immunoprecipitation show RAB40b and Kif16b complex together. Additionally, DCLK1, RAB40b, and Kif16b colocalize to invadopodia in confocal studies. We found that DCLK1 colocalizes with markers of mature invadopodia, and that DCLK1 colocalizes to Rab40b, Kif16b, and MMP9 within invadopodia. DCLK1 and MMP9 co-localize to invadopodia as well. Cells expressing DCLK1 had increased MMP 1, and 9 (p <0.041, p <0.001) expression compared to shDCLK1 cells. Further, shDCLK1 cells have significantly attenuated MMP 1 and 9 activity as measured by gelatin zymography. These studies reveal a novel role of DCLK1 in the molecular mechanism driving invadopodia, MMP trafficking and signaling. These early studies indicate that targeting DCLK1 in HNSCC may reduce locoregional invasion and help prevent early metastatic spread. Citation Format: Levi K. Arnold, David Standing, Prabhu Ramamoorthy, Harmony Saunders, Thuc Ly, Shrikant Anant, Sufi Thomas. Understanding the role of DCLK1 mediated invadopodia regulation in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2419.

Chemical Biology of Mitotic Spindle Assembly Motors.

Mitotic kinesins play essential roles during mitotic spindle assembly and in ensuring proper chromosome segregation. Chemical inhibitors of mitotic kinesins are therefore valuable tools to study kinesin function in vitro and in cells. Because cancer is a disease of unregulated cell division, inhibitors also represent potential chemotherapeutic agents. Here, we present assays that can be used to evaluate the potency and specificity of mitotic kinesin inhibitors identified from high-throughput screening. By evaluating their effects in a variety of in vitro, fixed-cell, and live cell assays, screening hits can be prioritized and optimized to produce effective, on-target inhibitors.

Understanding how kinesin motor proteins regulate postsynaptic function in neuron.

The Kinesin superfamily proteins (KIFs) are major molecular motors that transport diverse set of cargoes along microtubules to both the axon and dendrite of a neuron. Much of our knowledge about kinesin function is obtained from studies on axonal transport. Emerging evidence reveals how specific kinesin motor proteins carry cargoes to dendrites, including proteins, mRNAs and organelles that are crucial for synapse development and plasticity. In this review, we will summarize the major kinesin motors and their associated cargoes that have been characterized to regulate postsynaptic function in neuron. We will also discuss how specific kinesins are selectively involved in the development of excitatory and inhibitory postsynaptic compartments, their regulation by post-translational modifications (PTM), as well as their roles beyond conventional transport carrier.

Dynein- and kinesin- mediated intracellular transport on microtubules facilitates RABV infection

Rabies, caused by rabies virus (RABV), is one of the most important neurotropic zoonoses and poses a severe threat to human and animal health. Exploration of its mechanism of neural transmission is meaningful but still insufficient. Here, we described the effects of microtubule-depolymerizing drugs and inhibitors of microtubule motor proteins on RABV infection. Colchicine, a microtubule-depolymerizing drug, significantly impeded RABV production in N2a cells. Overexpression of CC1 or p50 attenuated viral infection through the functional disruption of cytoplasmic dynein, which was consistent with the inhibitory effect of Na3VO4, a dynein activity inhibitor. Moreover, transfection with Flag-KHCct impaired RABV infection, as cytoplasmic kinesin-based motility was blocked. These results demonstrated that RABV can infect N2a cells in a manner that depends on microtubule integrity as well as dynein and kinesin function.

Molecular characterization, dynamic transcription, and potential function of KIF3A/KIF3B during spermiogenesis in Opsariichthys bidens.

Spermiogenesis is the final phase of spermatogenesis, wherein the spermatids differentiate into mature spermatozoa via complex morphological transformation. In this process, kinesin plays an important role. Here, we observed the morphological transformation of spermatids and analyzed the characterization, dynamic transcription, and potential function of kinesin KIF3A/KIF3B during spermiogenesis in Chinese hook snout carp (Opsariichthys bidens). We found that the full-length cDNAs of O. bidens kif3a and kif3b were 2544 and 2806bp in length comprising 119bp and 259bp 5' untranslated region (UTR), 313bp and 222bp 3' UTR, and 2112bp and 2325bp open reading frame encoding 703 and 774 amino acids, respectively. Ob-KIF3A/KIF3B proteins have three domains, namely N-terminal head, coiled-coil stalk, and C-terminal tail, and exhibit high similarity with homologous proteins in vertebrates and invertebrates. Ob-kif3a/kif3b mRNAs were ubiquitously expressed in all tissues examined, with the highest expression in the brain and stage-IV testis. Immunofluorescence results showed that Ob-KIF3A was co-localized with tubulin and the mitochondria. Particularly, in early spermatids, Ob-KIF3A, tubulin, and the mitochondrial signals were evenly distributed in the cytoplasm, whereas in middle spermatids, they were distributed around the nucleus. In the late stage, the signals were concentrated on one side of the nucleus, where the tail is formed, whereas in mature sperms, they were detected in the midpiece and flagellum. These results indicate that Ob-KIF3A/KIF3B may participate in nuclear reshaping, flagellum formation, and mitochondrial aggregation in the midpiece during spermiogenesis.

Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation.

SUMMARYSynaptic structural plasticity, key to long-term memory storage, requires translation of localized RNAs delivered by long-distance transport from the neuronal cell body. Mechanisms and regulation of this system remain elusive. Here, we explore the roles of KIF5C and KIF3A, two members of kinesin superfamily of molecular motors (Kifs), and find that loss of function of either kinesin decreases dendritic arborization and spine density whereas gain of function of KIF5C enhances it. KIF5C function is a rate-determining component of local translation and is associated with ~650 RNAs, including EIF3G, a regulator of translation initiation, and plasticity-associated RNAs. Loss of function of KIF5C in dorsal hippocampal CA1 neurons constrains both spatial and contextual fear memory, whereas gain of function specifically enhances spatial memory and extinction of contextual fear. KIF5C-mediated long-distance transport of local translation substrates proves a key mechanism underlying structural plasticity and memory.

Roles of kinesin superfamily proteins in colorectal cancer carcinogenesis (Review).

Colorectal cancer (CRC), a commonly occurring carcinoma, now ranks the second in terms of cancer‑associated deaths around the world. Among the numerous factors that contribute to CRC tumor progression, a class of motor proteins known as the kinesins has been found to play a vital role. Kinesins are responsible for the intracellular trafficking of functional proteins, organelles and biomacromolecules along microtubules. Dysregulation of kinesins has been revealed to influence the cell cycle to cause abnormal cell growth and affect cell adhesion to promote epithelial‑mesenchymal transition in breast, bladder, ovarian and prostate cancer. Studies on the function of kinesins in CRC have also been performed, although, to the best of our knowledge, little is known about the underlying mechanisms of kinesins in CRC progression. The present review outlines the roles played by different kinesins in CRC carcinogenesis, mainly discussing the most studied subfamilies (kinesin3‑6, 8, 10, 11 and 13), This review aims to illustrate the functions of kinesins in CRC cell growth, cancer metastasis and chemoresistance to provide insights regarding kinesins as potential targets for determining CRC prognosis and selecting therapy.

A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health.

KIF1A is a critical cargo transport motor within neurons. More than 100 known mutations result in KIF1A-associated neurological disorder (KAND), a degenerative condition for which there is no cure. A missense mutation, P305L, was identified in children diagnosed with KAND, but the molecular basis for the disease is unknown. We find that this conserved residue is part of an unusual 310 helix immediately adjacent to the family-specific K-loop, which facilitates a high microtubule-association rate. We find that the mutation negatively affects several biophysical parameters of the motor. However, the microtubule-association rate of the motor is most markedly affected, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that the 310 helix facilitates a specific K-loop conformation that is critical for its function. We find that the function of this proline is conserved in kinesin-1, revealing a fundamental principle of the kinesin motor mechanism.

A Tug-of-War Model Explains the Saltatory Sperm Cell Movement in Arabidopsis thaliana Pollen Tubes by Kinesins With Calponin Homology Domain.

Upon pollination, two sperm cells are transported inside the growing pollen tube toward the apex. One sperm cell fertilizes the egg cell to form the zygote, while the other fuses with the two polar nuclei to form the triploid endosperm. In Arabidopsis thaliana, the transport of the two sperm cells is characterized by sequential forward and backward movements with intermediate pauses. Until now, it is under debate which components of the plant cytoskeleton govern this mechanism. The sperm cells are interconnected and linked to the vegetative nucleus via a cytoplasmic projection, thus forming the male germ unit. This led to the common hypothesis that the vegetative nucleus is actively transported via myosin motors along actin cables while pulling along the sperm cells as passive cargo. In this study, however, we show that upon occasional germ unit disassembly, the sperm cells are transported independently and still follow the same bidirectional movement pattern. Moreover, we found that the net movement of sperm cells results from a combination of both longer and faster runs toward the pollen tube apex. We propose that the observed saltatory movement can be explained by the function of kinesins with calponin homology domain (KCH). This subgroup of the kinesin-14 family actively links actin filaments and microtubules. Based on KCH's specific properties derived from in vitro experiments, we built a tug-of-war model that could reproduce the characteristic sperm cell movement in pollen tubes.

Phosphorylation of KIF18A Alters Its Mitotic Function and Localization

Kinesin motor proteins play important roles in intracellular transport, cell division, and cilia function. Eukaryotic cells express many kinesins (45 in mammals) to support these diverse cellular processes in cell cycle and cell type dependent manners. Of the 45 mammalian kinesins, 44 of them have identified sites of post-translational modification (PTM) within their enzymatic region, suggesting the activity of kinesins may be reversibly regulated. Our long-term goal is to investigate how kinesin function is controlled through these PTMs. We are currently investigating the effects of three previously identified phosphorylation sites (S244, S284, and S357) on the mitotic functions of KIF18A. KIF18A (kinesin-8) is a mammalian kinesin that suppresses the dynamics of microtubules to promote chromosome alignment during cell division. KIF18A is also known to play a role in kinetochore microtubule attachment and the metaphase to anaphase transition in some cell types. However, little is understood about how KIF18A is regulated to perform these various functions. Our data suggest that phosphorylation at S357 promotes the localization of KIF18A to non-kinetochore microtubules within the spindle. Additionally, mutation of S284 leads to accumulation of KIF18A at centrosomes, while a non-phosphorylatable substitution at S244 increases the time required to complete mitosis. Taken together this work indicates that phosphorylation of residues within KIF18A's enzymatic region are important for controlling its activity and localization during cell division.

Double Duty: Mitotic Kinesins and Their Post-Mitotic Functions in Neurons.

Neurons, regarded as post-mitotic cells, are characterized by their extensive dendritic and axonal arborization. This unique architecture imposes challenges to how to supply materials required at distal neuronal components. Kinesins are molecular motor proteins that mediate the active delivery of cellular materials along the microtubule cytoskeleton for facilitating the local biochemical and structural changes at the synapse. Recent studies have made intriguing observations that some kinesins that function during neuronal mitosis also have a critical role in post-mitotic neurons. However, we know very little about the function and regulation of such kinesins. Here, we summarize the known cellular and biochemical functions of mitotic kinesins in post-mitotic neurons.

AAV-CRB2 protects against vision loss in an inducible CRB1 retinitis pigmentosa mouse model

Loss of Crumbs homolog 1 (CRB1) or CRB2 proteins in Müller cells or photoreceptors in the mouse retina results in a CRB dose-dependent retinal phenotype. In this study, we present a novel Müller cell-specific Crb1KOCrb2LowMGC retinitis pigmentosa mouse model (complete loss of CRB1 and reduced levels of CRB2 specifically in Müller cells). The Crb double mutant mice showed deficits in electroretinography, optokinetic head tracking, and retinal morphology. Exposure of retinas to low levels of dl-α-aminoadipate acid induced gliosis and retinal disorganization in Crb1KOCrb2LowMGC retinas but not in wild-type or Crb1-deficient retinas. Crb1KOCrb2LowMGC mice showed a substantial decrease in inner/outer photoreceptor segment length and optokinetic head-tracking response. Intravitreal application of rAAV vectors expressing human CRB2 (hCRB2) in Müller cells of Crb1KOCrb2LowMGC mice subsequently exposed to low levels of dl-α-aminoadipate acid prevented loss of vision, whereas recombinant adeno-associated viral (rAAV) vectors expressing human CRB1 (hCRB1) did not. Both rAAV vectors partially protected the morphology of the retina. The results suggest that hCRB expression in Müller cells is vital for control of retinal cell adhesion at the outer limiting membrane, and that the rAAV-cytomegalovirus (CMV)-hCRB2 vector is more potent than rAAV-minimal CMV (CMVmin)-hCRB1 in protection against loss of vision.

Role of dynein-dynactin complex, kinesins, motor adaptors, and their phosphorylation in dendritogenesis.

One of the characteristic features of different classes of neurons that is vital for their proper functioning within neuronal networks is the shape of their dendritic arbors. To properly develop dendritic trees, neurons need to accurately control the intracellular transport of various cellular cargo (e.g., mRNA, proteins, and organelles). Microtubules and motor proteins (e.g., dynein and kinesins) that move along microtubule tracks play an essential role in cargo sorting and transport to the most distal ends of neurons. Equally important are motor adaptors, which may affect motor activity and specify cargo that is transported by the motor. Such transport undergoes very dynamic fine-tuning in response to changes in the extracellular environment and synaptic transmission. Such regulation is achieved by the phosphorylation of motors, motor adaptors, and cargo, among other mechanisms. This review focuses on the contribution of the dynein-dynactin complex, kinesins, their adaptors, and the phosphorylation of these proteins in the formation of dendritic trees by maturing neurons. We primarily review the effects of the motor activity of these proteins in dendrites on dendritogenesis. We also discuss less anticipated mechanisms that contribute to dendrite growth, such as dynein-driven axonal transport and non-motor functions of kinesins.

Applying the Brakes: Understanding the Conformational Changes in Kinesin‐5

Human Kinesin‐5 (Eg5), an anticancer drug target, is key to the assembly of the bipolar spindle during mitosis. Its promise as a clinical target comes from the fact that it is involved only in metaphase and not interphase like some of the leading clinical drugs; however, the overall mechanism of inhibition needs further exploration. Inspection of the crystal structures of Kinesin‐1 dimers reveal the addition of strands to the central β‐sheet is found in one head and absent in the other; this previously unrecognized structural asymmetry between the two heads may suggest a regulatory mechanism involving the N‐terminal Neck‐Linker (NL) and the C‐Terminal Cover‐Neck. These structural changes in the neck‐linker and cover‐neck of Kinesin‐5 have not been reported. Thus, the importance of establishing this conformational switch in Kinesin‐5 requires additional experiments for understanding and molecular validation. However, we must first understand how changes in the neck‐linker modulate kinesin function. We have made mutations at the start of the neck‐linker of dimeric Kinesin‐5 (1‐513) and KHC (1‐560). Our results indicate these neck‐linker mutants alter kinesin ATPase activity. This indicates that the neck‐linker participates in allosteric regulation of kinesin. Our future goal is to test the response to inhibition by current anticancer inhibitors such as S‐trityl‐L‐cysteine.Support or Funding InformationBUILD Technicians supported under grant #RL5GM118966 and BUILD undergraduate students under grant #TL4GM118968. An Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #5 P20 GM103424‐15 and 3 P20 GM103424‐15S1. The Louisiana Cancer Research Consortium (LCRC) and National Center for Research Resources RCMI program Grant #8 G12MD007595 and the Louisiana Biomedical Research Network Grant #8P20GM103424 The National Institute of Minority Health and Health Disparities of the National Institutes of Health under award #G12MD007595. Students are supported by GRAD Star Scholar Mini Grants through Center for Undergraduate Research and Graduate Opportunity (CURGO).