RESUMEN
Kinesin-1 and Growth Associated Protein 43 (GAP-43) localization in muscle fiber are crucial for proper skeletal muscle hypertrophy. To evaluate this assumption, we investigated the beneficial effects of endurance training on GAP-43 and Kinesin Family Member 5B (KIF5B) expression in gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Fifty-two male rats were randomly divided into four groups: healthy control (C), healthy trained (T), diabetic control (DC) and diabetic trained (DT). Diabetes was induced by a single intraperitoneal injection of STZ (45 mg/kg). The rats in DT and T groups were subjected to treadmill running for 5 days a week over 6 weeks. The results indicated that the GAP-43 and KIF5B protein levels in the DC group were significantly lower than those in the C group. Additionally, chronic treadmill running in diabetic rats was accompanied by significant increase of GAP-43 and KIF5B protein expression, compared to DC group. Furthermore, the endurance training in healthy rats was associated with a significant increase of GAP-43 and KIF5B protein levels. In addition, we found positive correlation between GAP-43 and KIF5B protein levels and myonuclear number per fiber and average gastrocnemius cross-sectional area (CSA). GAP43 and KIF5B protein levels were decreased in skeletal muscles of diabetic rats, and exercise training had beneficial effects and could restore their abnormal expression. Moreover, there is a strong relationship between muscle hypertrophy and GAP-43 and KIF5B protein levels.
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Diabetes Mellitus Experimental/patología , Proteína GAP-43/análisis , Cinesinas/análisis , Fibras Musculares Esqueléticas/patología , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Entrenamiento Aeróbico , Proteína GAP-43/metabolismo , Cinesinas/metabolismo , Masculino , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Condicionamiento Físico Animal , Ratas WistarRESUMEN
Liver cancer is thought as the most common human malignancy worldwide, and hepatocellular carcinoma (HCC) accounts for nearly 90% liver cancer. Due to its poor early diagnosis and limited treatment, HCC has therefore become the most lethal malignant cancers in the world. Recently, molecular targeted therapies showed great promise in the treatment of HCC, and novel molecular therapeutic targets is urgently needed. KIF15 is a microtubule-dependent motor protein involved in multiple cell processes, such as cell division. Additionally, KIF15 has been reported to participate in the growth of various types of tumors; however, the relation between KIF15 and HCC is unclear. Herein, our study investigated the possible role of KIF15 on the progression of HCC and found that KIF15 has high expression in tumor samples from HCC patients. KIF15 could play a critical role in the regulation of cell proliferation of HCC, which was proved by in vitro and in vivo assays. In conclusion, this study confirmed that KIF15 could be a novel therapeutic target for the treatment of HCC.
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Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/patología , Cinesinas/metabolismo , Neoplasias Hepáticas/patología , Adulto , Anciano , Animales , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Cinesinas/análisis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana EdadRESUMEN
Molecular motors play important roles in force generation, migration, and intracellular trafficking. Changes in specific motor activities are altered in numerous diseases. KIF20A, a motor protein of the kinesin-6 family, is overexpressed in bladder cancer, and KIF20A levels correlate negatively with clinical outcomes. We report here a new role for the KIF20A kinesin motor protein in intracellular mechanics. Using optical tweezers to probe intracellular mechanics and surface AFM to probe cortical mechanics, we first confirm that bladder urothelial cells soften with an increasing cancer grade. We then show that inhibiting KIF20A makes the intracellular environment softer for both high- and low-grade bladder cancer cells. Upon inhibition of KIF20A, cortical stiffness also decreases in lower grade cells, while it surprisingly increases in higher grade malignant cells. Changes in cortical stiffness correlate with the interaction of KIF20A with myosin IIA. Moreover, KIF20A inhibition negatively regulates bladder cancer cell motility irrespective of the underlying substrate stiffness. Our results reveal a central role for a microtubule motor in cell mechanics and migration in the context of bladder cancer.
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Cinesinas/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Fenómenos Biomecánicos , Línea Celular Tumoral , Movimiento Celular , Humanos , Cinesinas/análisis , Miosinas/análisis , Miosinas/metabolismo , Pinzas Ópticas , Reología , Vejiga Urinaria/citología , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
The arrangement of immature germ cells changes regularly and periodically along the axis of the seminiferous tubule, and is used to describe the progression of spermatogenesis. This description is based primarily on the changes in the acrosome and the nuclear morphology of haploid spermatids. However, such criteria cannot be applied under pathological conditions with arrested spermatid differentiation. In such settings, the changes associated with the differentiation of premeiotic germ cells must be analyzed. Here, we found that the unique bipolar motor protein, KIF11 (kinesin-5/Eg5), which functions in spindle formation during mitosis and meiosis in oocytes and early embryos, is expressed in premeiotic germ cells (spermatogonia and spermatocytes). Thus, we aimed to investigate whether KIF11 could be used to describe the progression of incomplete spermatogenesis. Interestingly, KIF11 expression was barely observed in haploid spermatids and Sertoli cells. The KIF11 staining allowed us to evaluate the progression of meiotic processes, by providing the time axis of spindle formation in both normal and spermatogenesis-arrested mutant mice. Accordingly, KIF11 has the potential to serve as an excellent marker to describe spermatogenesis, even in the absence of spermatid development.
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Cinesinas/análisis , Túbulos Seminíferos/citología , Espermatogénesis , Animales , Masculino , Meiosis , Ratones , Ratones Endogámicos C57BL , Túbulos Seminíferos/ultraestructura , Espermátides/citología , Espermatocitos/citología , Espermatogonias/citologíaRESUMEN
Covalent labeling with mass spectrometry (CL-MS) provides a direct measure of the chemical and structural features of proteins with the potential for resolution at the amino-acid level. Unfortunately, most applications of CL-MS are limited to narrowly defined differential analyses, where small numbers of residues are compared between two or more protein states. Extending the utility of high-resolution CL-MS for structure-based applications requires more robust computational routines and the development of methodology capable of reporting of labeling yield accurately. Here, we provide a substantial improvement in the analysis of CL-MS data with the development of an extended plug-in built within the Mass Spec Studio development framework (MSS-CLEAN). All elements of data analysis-from database search to site-resolved and normalized labeling output-are accommodated, as illustrated through the nonselective labeling of the human kinesin Eg5 with photoconverted 3,3'-azibutan-1-ol. In developing the new features within the CL-MS plug-in, we identified additional complexities associated with the application of CL reagents, arising primarily from digestion-induced bias in yield measurements and ambiguities in site localization. A strategy is presented involving the use of redundant site labeling data from overlapping peptides, the imputation of missing data, and a normalization routine to determine relative protection factors. These elements together provide for a robust structural interpretation of CL-MS/MS data while minimizing the over-reporting of labeling site resolution. Finally, to minimize bias, we recommend that digestion strategies for the generation of useful overlapping peptides involve the application of complementary enzymes that drive digestion to completion.
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Marcaje Isotópico/métodos , Cinesinas/análisis , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Humanos , Cinesinas/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Traumatic brain injury (TBI) is characterized by various micro- and macrostructural neuropathological changes which can be identified in the light microscope examination. The most common pathophenotype of TBI visualized in postmortem neuropathological assessment includes neuron injury with involvement of all of its structural regions followed by its progressive degeneration defined as traumatic axonal injury (TAI). This is directly related with disruption of the axolemmal cytoskeletal network architecture resulting in breakdown, dissolution and accumulation of a number of neuronal proteins. Regarding the availability and progress in the development of specific antibodies against neuronal proteins, their usage is restricted due to low specificity for injured axons in the pathomechanism of TBI followed by TAI. Taking this into account with relation to expanding the role of axonal cytoskeleton and its based biomarkers we have presented a study documenting neuropathological features concerning the expression of dynein (DNAH9), dynactin (DCTN1) and kinesin (KIF5B) in the brain specimens obtained during forensic autopsies from TBI victims. The study was carried out using cases (n = 21) of severe head injury suspected to be the cause of death and control cases (n = 17) of sudden death in the mechanism of cardiopulmonary failure along with a positive control case which died after suicidal gunshot injury. In our study, we documented that DNAH9, DCTN1, and KIF5B staining should be considered as a supplemental diagnostic tool for TBI in postmortem neuropathological examination and forensic autopsy. This additional motor protein immunohistochemical staining procedure could be useful in the evaluation of lesions that may remain undiagnosed during a routine examination and aid in more accurate identification of TBI followed by TAI.
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Autopsia/métodos , Dineínas Axonemales/análisis , Lesiones Traumáticas del Encéfalo/diagnóstico , Complejo Dinactina/análisis , Cinesinas/análisis , Biomarcadores/análisis , Encéfalo/metabolismo , Encéfalo/patología , Patologia Forense/métodos , HumanosRESUMEN
Axons in the central nervous system (CNS) typically fail to regenerate after injury. This failure is multi-factorial and caused in part by disruption of the axonal cytoskeleton. The cytoskeleton, in particular microtubules (MT), plays a critical role in axonal transport and axon growth during development. In this regard, members of the kinesin superfamily of proteins (KIFs) regulate the extension of primary axons toward their targets and control the growth of collateral branches. KIF2A negatively regulates axon growth through MT depolymerization. Using three different injury models to induce SCI in adult rats, we examined the temporal and cellular expression of KIF2A in the injured spinal cord. We observed a progressive increase of KIF2A expression with maximal levels at 10 days to 8 weeks post-injury as determined by Western blot analysis. KIF2A immunoreactivity was present in axons, spinal neurons and mature oligodendrocytes adjacent to the injury site. Results from the present study suggest that KIF2A at the injured axonal tips may contribute to neurite outgrowth inhibition after injury, and that its increased expression in inhibitory spinal neurons adjacent to the injury site might contribute to an intrinsic wiring-control mechanism associated with neuropathic pain. Further studies will determine whether KIF2A may be a potential target for the development of regeneration-promoting or pain-preventing therapies.
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Cinesinas/análisis , Cinesinas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Axones/metabolismo , Axones/patología , Modelos Animales de Enfermedad , Cinesinas/genética , Masculino , Regeneración Nerviosa/genética , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patologíaRESUMEN
The chromokinesin Kif4A controls proper chromosome condensation, congression/alignment, and cytokinesis to ensure faithful genetic inheritance. Here, we report that Cdk phosphorylation of human Kif4A at T1161 licenses Kif4A chromosomal localization, which, in turn, controls Kif4A early mitotic function. Phosphorylated Kif4A (Kif4AWT) or Cdk phospho-mimetic Kif4A mutant (Kif4ATE) associated with chromosomes and condensin I (non-SMC subunit CAP-G and core subunit SMC2) to regulate chromosome condensation, spindle morphology, and chromosome congression/alignment in early mitosis. In contrast, Cdk non-phosphorylatable Kif4A mutant (Kif4ATA) could neither localize on chromosomes nor associate with CAP-G and SMC2. Furthermore, Kif4ATA could not rescue defective chromosome condensation, spindle morphology, or chromosome congression/alignment in cells depleted of endogenous Kif4A, which activated a mitotic checkpoint and delayed early mitotic progression. However, targeting Kif4ATA to chromosomes by fusion of Kif4ATA with Histone H1 resulted in restoration of chromosome and spindle functions of Kif4A, similar to Kif4AWT and Kif4ATE, in cells depleted of endogenous Kif4A. Thus, our results demonstrate that Cdk phosphorylation-licensed chromosomal localization of Kif4A plays a critical role in regulating early mitotic functions of Kif4A that are important for early mitotic progression.
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Quinasas Ciclina-Dependientes/metabolismo , Cinesinas/metabolismo , Mitosis , Adenosina Trifosfatasas/análisis , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromosomas Humanos/metabolismo , Cromosomas Humanos/ultraestructura , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Cinesinas/análisis , Modelos Moleculares , Complejos Multiproteicos/análisis , Complejos Multiproteicos/metabolismo , Fosforilación , Huso Acromático/metabolismo , Huso Acromático/ultraestructuraRESUMEN
With recent advances in understanding the genomic underpinnings and oncogenic drivers of pathogenesis in different subtypes, it is increasingly clear that proper pretreatment diagnostics are essential for the choice of appropriate treatment options for non-small cell lung cancer (NSCLC). Tumor tissue preservation in optimal cutting temperature (OCT) compound is commonly used in the surgical suite. However, proteins recovered from OCT-embedded specimens pose a challenge for LC-MS/MS experiments, due to the large amounts of polymers present in OCT. Here we present a simple workflow for whole proteome analysis of OCT-embedded NSCLC tissue samples, which involves a simple trichloroacetic acid precipitation step. Comparisons of protein recovery between frozen versus OCT-embedded tissue showed excellent consistency with more than 9200 proteins identified. Using an isobaric labeling strategy, we quantified more than 5400 proteins in tumor versus normal OCT-embedded core needle biopsy samples. Gene ontology analysis indicated that a number of proliferative as well as squamous cell carcinoma (SqCC) marker proteins were overexpressed in the tumor, consistent with the patient's pathology based diagnosis of "poorly differentiated SqCC". Among the most downregulated proteins in the tumor sample, we noted a number of proteins with potential immunomodulatory functions. Finally, interrogation of the aberrantly expressed proteins using a candidate approach and cross-referencing with publicly available databases led to the identification of potential druggable targets in DNA replication and DNA damage repair pathways. We conclude that our approach allows LC-MS/MS proteomic analyses on OCT-embedded lung cancer specimens, opening the way to bring powerful proteomics into the clinic. Graphical Abstract á .
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Biopsia con Aguja Gruesa , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos , Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Cromatografía Liquida , Regulación Neoplásica de la Expresión Génica , Humanos , Cinesinas/análisis , Cinesinas/genética , Cinesinas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodos , Reproducibilidad de los Resultados , Relación Señal-Ruido , TemperaturaRESUMEN
Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins â¼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, â¼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.
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Compuestos de Cadmio/química , Cinesinas/análisis , Imagen Óptica/métodos , Puntos Cuánticos/química , Compuestos de Selenio/química , Sulfuros/química , Células HeLa , Humanos , Ligandos , Nanotecnología , Agua/químicaRESUMEN
Medulloblastoma (MB) is the most common malignant brain tumor in childhood. At present, there is no well-established targeted drug for majority of patients. The kinesin family member 14 (KIF14) is a novel oncogene located on chromosome 1q and is dysregulated in multiple cancers. The objectives of this study were to evaluate KIF14 expression and chromosome 1q copy number in MB, and to delineate its biological functions in MB pathogenesis. By quantitative RT-PCR and immunohistochemistry, we found KIF14 was overexpressed in MB. Increased KIF14 expression at protein level was strongly associated with shorter progression-free survival (P=0.0063) and overall survival (P=0.0083). Fluorescence in situ hybridization (FISH) analysis confirmed genomic gain of chromosome 1q in 17/93 (18.3%) of MB. Combined genetic and immunohistochemical analyses revealed that 76.5% of MB with 1q gain showed consistent overexpression of KIF14, and a tight link between chromosome 1q gain and KIF14 overexpression (P=0.03). Transient, siRNAs-mediated downregulation of KIF14 suppressed cell proliferation and induced apoptosis in two MB cell lines. Stably KIF14 knockdown by shRNAs inhibited cell viability, colony formation, migration and invasion, and tumor sphere formation in MB cells. We conclude that KIF14 is dysregulated in MB and is an adverse prognostic factor for survival. Furthermore, KIF14 is part of MB biology and is a potential therapeutic target for MB.
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Apoptosis/genética , Regulación hacia Abajo/genética , Cinesinas/genética , Cinesinas/metabolismo , Meduloblastoma/metabolismo , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Adolescente , Adulto , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Cinesinas/análisis , Masculino , Meduloblastoma/química , Proteínas Oncogénicas/análisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Adulto JovenRESUMEN
Engineering cargo-loading strategies is crucial to developing nanotechnological applications of microtubule-based biomolecular transport systems. Here, we report a highly efficient and robust bioconjugation scheme to load antibodies to microtubules. Our method takes advantage of the inverse-electron-demand Diels-Alder addition reaction between tetrazine and trans-cyclooctene: the fastest known bioorthogonal reaction, characterized by its excellent selectivity and biocompatibility. As proof of concept, we performed kinesin-1 gliding motility assays with antibody-conjugated microtubules and demonstrated the highly sensitive detection of fluorescent protein analyte down to 0.1 pM in microliter sample volumes. Importantly, the detection selectivity was retained in the presence of other fluorescent background proteins. We envision the applicability of our fast, simple, and robust conjugation method to a wide range of biosensing platforms based on biomolecular transport systems.
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Ciclooctanos/química , Colorantes Fluorescentes/química , Inmunoconjugados/química , Proteínas de Microtúbulos/análisis , Microtúbulos/química , Animales , Reacción de Cicloadición , Insectos , Cinesinas/análisis , Ratones , Microscopía Fluorescente , Modelos Moleculares , Tubulina (Proteína)/análisisRESUMEN
1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution.
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Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Cinesinas/antagonistas & inhibidores , Cinesinas/análisis , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacología , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Microscopía FluorescenteRESUMEN
To determine the prognostic significance of Kinesin family member 2C (KIF-2C) expression in patients with operable esophageal squamous cell carcinoma (ESCC), we conducted an immunohistochemical analysis of KIF-2C expression in 415 surgically resected primary tumor tissues and 40 adjacent non-cancerous tissues from patients with operable ESCC. The median duration of postoperative follow-up was 76.0 months. Higher KIF-2C expression was associated with significantly increased risks of higher pathologic tumor (pT) status (P=0.038) and poorer tumor differentiation (P=0.022). For the entire cohort, KIF-2C expression was not an independent factor significantly associated with overall survival (OS) (P=0.097) or disease-free survival (DFS) (P=0.152). In female patients, KIF-2C expression had no effect on OS (P=0.880) and DFS (P=0.864). However, OS (hazard ratio (HR)=1.480, P=0.013) and DFS (HR=1.418, P=0.024) were worse for male patients with high KIF-2C expression compared with male patients with low KIF-2C expression. Moreover, the OS and DFS of male patients with high KIF-2C expression were also significantly shorter compared with female patients with low KIF-2C expression (P=0.022, P=0.029) and female patients with high KIF-2C expression (P=0.014, P=0.018). Based on these findings, KIF-2C expression in tumor tissues promises to serve as an independent prognostic marker for male, but not female, patients with operable ESCC. Prognosis was worse for male patients with high KIF-2C expression compared with patients with the same pathologic tumor-node-metastasis (pTNM) stage.
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Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/cirugía , Neoplasias Esofágicas/química , Neoplasias Esofágicas/cirugía , Esofagectomía , Cinesinas/análisis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Diferenciación Celular , Supervivencia sin Enfermedad , Neoplasias Esofágicas/mortalidad , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Esofagectomía/efectos adversos , Esofagectomía/mortalidad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Factores Sexuales , Factores de Tiempo , Resultado del Tratamiento , Regulación hacia ArribaRESUMEN
Microtubules (MTs) are cylindrical polymers of αß-tubulin that display pseudo-helical symmetry due to the presence of a lattice seam of heterologous lateral contacts. The structural similarity between α- and ß-tubulin makes it difficult to computationally distinguish them in the noisy cryo-EM images, unless a marker protein for the tubulin dimer, such as kinesin motor domain, is present. We have developed a new data processing protocol that can accurately determine αß-tubulin register and seam location for MT segments. Our strategy can deal with difficult situations, where the marker protein is relatively small or the decoration of marker protein is sparse. Using this new seam-search protocol, combined with movie processing for data from a direct electron detection camera, we were able to determine the cryo-EM structures of MT at 3.5 Å resolution in different functional states. The successful distinction of α- and ß-tubulin allowed us to visualize the nucleotide state at the E-site and the configuration of lateral contacts at the seam.
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Microscopía por Crioelectrón/métodos , Cinesinas/análisis , Proteínas Asociadas a Microtúbulos/análisis , Microtúbulos/ultraestructura , Tubulina (Proteína)/análisis , Humanos , Modelos MolecularesRESUMEN
It has been shown that the dysfunction of N-methyl-d-asparate (NMDA) receptors-mediated neurotransmission plays a role in the pathophysiology of schizophrenia. Especially, GluN2B, a subunit of NMDA receptors, associated trafficking complex is altered in the prefrontal cortex of schizophrenia. The kinesin superfamily motor protein 17 (KIF17) is known as a transporter of NR2B.Previous studies showed that a structural variant of KIF17 gene is associated with a schizophrenic phenotype. Therefore, here we investigated KIF17 levels in postmortem prefrontal cortex in schizophrenia and the association of a missense polymorphism (Ile341Val) in KIF17 with schizophrenia. The protein expression of KIF17 in schizophrenic postmortem brains was significantly lower than that in controls. Next, the association of missense polymorphisms (rs631375, rs13375609, rs522496 and rs2296225) of KIF17 gene in 567 schizophrenia and 710 healthy subjects was examined. Both genotypic distribution and allelic frequency of rs2296225 polymorphism were significantly different between the chronic schizophrenia subjects and controls. However, our findings described above were not replicated with the independent subjects (555 schizophrenia and 814 healthy controls). Furthermore, the two alleles of rs2296225 polymorphism did not affect the mRNA expression of KIF17. These results suggest that the dysfunction of KIF17 might be involved in the pathophysiology of schizophrenia.
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Cinesinas/análisis , Mutación Missense , Polimorfismo de Nucleótido Simple , Corteza Prefrontal/metabolismo , Esquizofrenia/genética , Adulto , Autopsia , Femenino , Frecuencia de los Genes , Voluntarios Sanos , Humanos , Cinesinas/genética , Masculino , Persona de Mediana Edad , Transporte de Proteínas/genética , ARN Mensajero/metabolismo , Esquizofrenia/fisiopatologíaRESUMEN
Total internal reflection fluorescence microscopy (TIRFM) is a wide-field illumination technique that illuminates only the molecules near the glass coverslip. It has become widely used in biological imaging because it has a significantly reduced background and high temporal resolution capability. The principles of TIRFM are illustrated in this protocol, in which the movements of motor proteins are imaged as they move along microtubules within live axonemes.
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Dineínas/análisis , Procesamiento de Imagen Asistido por Computador/métodos , Cinesinas/análisis , Microscopía Fluorescente/métodos , Microtúbulos/química , Transporte de ProteínasRESUMEN
AIM: To compare kinesin family member 1B (KIF1B) expression with clinicopathologic parameters and prognosis in hepatocellular carcinoma (HCC) patients. METHODS: KIF1B protein and mRNA expression was assessed in HCC and paracarcinomatous (PC) tissues from 68 patients with HCC using Western blot and quantitative real-time reverse transcription-PCR, respectively. Student's t-tests were used to analyze relationships between clinicopathologic parameters and KIF1B expression, the Kaplan-Meier method was used to analyze survival outcomes, and the log-rank test was used to compare survival differences between groups. RESULTS: Mean protein and mRNA levels of KIF1B were similar between HCC and PC tissues. However, HCC tissues with vein invasions had significantly lower KIF1B protein levels compared to those without vein invasions (2.30 ± 0.82 relative units vs 2.77 ± 0.84 relative units, P < 0.05). KIF1B protein levels in HCC tissues from patients with recurrence during the follow-up period were significantly lower than those without recurrence (2.31 ± 0.92 relative units vs 2.80 ± 0.80 relative units, P < 0.05). However, KIF1B protein and mRNA expression in HCC patients was not associated with other clinicopathologic parameters. Ratios of KIF1B mRNA expression in HCC tissues to those in PC tissues were correlated with overall survival (13.5 mo vs 20.0 mo, P < 0.05) and disease-free survival (11.5 mo vs 19.5 mo, P < 0.05). CONCLUSION: Downregulation of KIF1B in HCC tissues is associated with poor prognosis; additional clinical studies are needed to confirm whether KIF1B can serve as a prognostic marker.
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Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Cinesinas/genética , Neoplasias Hepáticas/genética , ARN Mensajero/genética , Anciano , Biomarcadores de Tumor/análisis , Western Blotting , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Cinesinas/análisis , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new â¼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed â¼2 nm precision.
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Microscopía Fluorescente/métodos , Puntos Cuánticos/química , Anticuerpos de Dominio Único/química , Algoritmos , Membrana Celular/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Canales Iónicos/análisis , Canales Iónicos/genética , Canales Iónicos/metabolismo , Cinesinas/análisis , Cinesinas/metabolismo , Microscopía Electrónica de Transmisión , Microtúbulos/metabolismo , Sondas Moleculares/química , Neuronas/metabolismo , Receptores AMPA/análisis , Receptores AMPA/metabolismo , Anticuerpos de Cadena Única/químicaRESUMEN
Quantum dots (QDs) have found a wide range of biological applications as fluorophores due to their extraordinary brightness and high photostability that are far superior to those of conventional organic dyes. These traits are particularly appealing for studying cell biology under a cellular autofluorescence background and with a long observation period. However, it remains the most important open challenge to target QDs at native intracellular molecules and organelles in live cells. Endocytosis-based delivery methods lead to QDs encapsulated in vesicles that have their surface biorecognition element hidden from the intracellular environment. The probing of native molecules using QDs has been seriously hindered by the lack of consistent approaches for delivery of QDs with exposed surface groups. In this study, we demonstrate that electroporation (i.e., the application of short electric pulses for cell permeabilization) generates reproducible results for delivering QDs into cells. We show evidence that electroporation-based delivery does not involve endocytosis or vesicle encapsulation of QDs. The amount of QD loading and the resulting cell viability can be adjusted by varying the parameters associated with the electroporation operation. To demonstrate the application of our approach for intracellular targeting, we study single-molecule motility of kinesin in live cells by labeling native kinesins using electroporation-delivered QDs. We envision that electroporation may serve as a simple and universal tool for delivering QDs into cells to label and probe native molecules and organelles.