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Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health. [ABSTRACT FROM AUTHOR]

Abstract Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.

Background: Nor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive. Methods: We used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy. Results: Here, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals. Conclusions: Our study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

A series of amide derivatives of 1,3,4-thiadiazole-benzimidazole (13a-j) has been designed, synthesized and screened for their anticancer activities against human breast cancer cell line (MCF-7), human lung cancer (A549), human colon cancer (Colo-205) & human ovarian cancer (A2780) by employing of MTT method with Etoposide as standard drug. Among the synthesized derivatives, the compound 13a with electron donating 3,4,5-trimethoxy substituent showed potent anticancer activity (MCF-7 = 0.04 ± 0.008 µM; A549 = 0.06 ± 0.009 µM; Colo-205 = 0.12 ± 0.067 µM & A2780 = 0.19 ± 0.071 µM. The compound 13a, shown two H-bonding contacts with PHE106, TYR 67 amino acids are O and NH group, as well as with additional hydrophobic interactions, including − stacking with aromatic rings and also had a binding affinity of −12.04 kcal/mole.

A new library of Aryl Benzimidazole-Pyridones (9a–j) has been synthesized, their structures were determined by 1HNMR,13CNMR and mass spectral data. Further, these compounds explored for their anticancer application on four human cancer cell lines such as human prostate cancer cell line (PC3), lung cancer (A549), breast cancer (MCF-7) and human ovarian cancer (A2780) by employing MTT assay and results were compared with known anticancer agent asetoposide. All these compounds displayed remarkable cytotoxic activity. Among them, five compounds 9a, 9b, 9c, 9h and 9i displayed more potent activity. All compounds exhibited a discerning cytotoxic effect on cancer cells while not affecting normal Vero cells (IC50 = >22 µM), so providing justification for the strategic design approach employed in the development of a targeted anticancer drug. [ABSTRACT FROM AUTHOR]

The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule-binding proteins, we have developed an unbiased biochemical assay, which relies on the selective extraction of cytosolic proteins from U2OS cells, while leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule-stabilising drug taxol, which is quantitated by mass spectrometry. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule-binding proteins, from which we have selected the ubiquitin E3 ligase tripartite motif-containing protein 3 (TRIM3) for further characterisation. We map TRIM3 microtubule binding to its C-terminal NHL-repeat region. We show that TRIM3 is required for the accumulation of acetylated tubulin, following treatment with taxol. Furthermore, loss of TRIM3 partially recapitulates the reduction in nocodazole-resistant microtubules characteristic of a-tubulin acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 following depletion of TRIM3 that is independent of transcription. [ABSTRACT FROM AUTHOR]

Abstract Background The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia. Results In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF. Conclusions Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.

The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large "superclusters" wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered "emptying" of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

Background: The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia. Results: In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF. Conclusions: Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments. [ABSTRACT FROM AUTHOR]

Scanning probe microscopy is used to study the effect of cytotoxic agents on the biomechanical and physicochemical properties of tumor cells and tissues. The possibility of using quantitative data of atomic force and capillary microscopy in assessing the morphological characteristics of cells during carcinogenesis is considered. The prospects of using morphometric analysis in biomedicine, biomechanics, regenerative medicine, and drug testing are described. The roughness of the control sample of HeLa human cervical cancer cells in terms of the average (Ra) and root-mean-square values (Rq) was 35 ± 5/48 ± 6 nm upon subtraction of a spline and 33 ± 5/45 ± 6 nm upon subtraction of a parabola, respectively. The roughness remains almost the same under the impact of cisplatin whereas nocodazole significantly increases the roughness to 51 ± 5/68 ± 6 nm and 56 ± 6/72 ± 7 nm, respectively. [ABSTRACT FROM AUTHOR]

TonEBP is a transcription factor known for its involvement in diverse physiological processes, including cell cycle, mitosis, migration, and cytoskeletal remodeling. However, the role of TonEBP regarding microtubules, essential structural components of the cytoskeleton, remains unclear. Here, we introduce a novel function for TonEBP as a regulator of microtubule nucleation. Our initial findings reveal that Nocodazole, a well-known microtubule depolymerizing agent, significantly downregulates the protein level of TonEBP. Moreover, microtubule depolymerization induces rapid degradation of TonEBP through the ubiquitin-proteasome pathway. Knockdown of TonEBP results in enhanced microtubule polymerization and regrowth, whereas the presence of TonEBP impairs microtubule nucleation. Collectively, our data suggest that TonEBP negatively regulates microtubule nucleation. • Microtubule inhibiting agents trigger rapid degradation of TonEBP. • Microtubule cytoskeleton is essential for TonEBP stability. • TonEBP acts as an inhibitor of microtubule nucleation at the centrosome. • TonEBP degradation is necessary for the regrowth of microtubules from the centrosome. [ABSTRACT FROM AUTHOR]

Wallerian degeneration, the progressive disintegration of distal axons and myelin that occurs after peripheral nerve injury, is essential for creating a permissive microenvironment for nerve regeneration, and involves cytoskeletal reconstruction. However, it is unclear whether microtubule dynamics play a role in this process. To address this, we treated cultured sciatic nerve explants, an in vitro model of Wallerian degeneration, with the microtubule-targeting agents paclitaxel and nocodazole. We found that paclitaxel-induced microtubule stabilization promoted axon and myelin degeneration and Schwann cell dedifferentiation, whereas nocodazole-induced microtubule destabilization inhibited these processes. Evaluation of an in vivo model of peripheral nerve injury showed that treatment with paclitaxel or nocodazole accelerated or attenuated axonal regeneration, as well as functional recovery of nerve conduction and target muscle and motor behavior, respectively. These results suggest that microtubule dynamics participate in peripheral nerve regeneration after injury by affecting Wallerian degeneration. This study was approved by the Animal Care and Use Committee of Southern Medical University, China (approval No. SMU-L2015081) on October 15, 2015.

Eukaryotic cells rely on long-lived microtubules for intracellular transport and as compression-bearing elements. We considered that long-lived microtubules are acetylated inside their lumen and that microtubule acetylation may modify microtubule mechanics. Here, we found that tubulin acetylation is required for the mechanical stabilization of long-lived microtubules in cells. Depletion of the tubulin acetyltransferase TAT1 led to a significant increase in the frequency of microtubule breakage. Nocodazole-resistant microtubules lost upon removal of acetylation were largely restored by either pharmacological or physical removal of compressive forces. In in vitro reconstitution experiments, acetylation was sufficient to protect microtubules from mechanical breakage. Thus, acetylation increases mechanical resilience to ensure the persistence of long-lived microtubules.

Microtubule targeting agents (MTAs) are the potential drug candidates for anticancer drug discovery. Disrupting the microtubule formation or inhibiting the de‐polymerization process by a synthetic molecule can lead to an excellent anticancer drug candidate. Here, we present the 2,5‐substituted‐1H‐benzo[d]imidazole derivatives as potential colchicine, nocodazole binding site targeting agents. About 20 benzimidazole derivatives were synthesized with 82.0%–94.0% yield using mild reaction conditions. The synthesized compounds showed moderate to excellent anticancer activity established in three cell lines, including Hela cells, A549 cells, MRC‐5 cells. The compounds B15, B16, B19, and B20 are the potential candidates with the IC50 values <15 μM in the three different cell lines. In MTT assay, compounds B15, B16, B19, and B20 showed excellent antiproliferation activity indicated by IC50 values in the range of 5.3 ± 0.21 to 18.1 ± 0.32 μM using HeLa and A549 cell lines. The predicted absorption, distribution, metabolism and excretion (ADME) properties and drug‐likeness properties of B15, B16, B19, and B20 indicate that these compounds can be used as lead compounds for further study to develop excellent MTAs. [ABSTRACT FROM AUTHOR]

The first development method of brewery shochu yeast focusing on chromosomal aneuploidy is reported in this study. Euploidy diploid shochu yeast S-3 was treated with a microtubule inhibitor, nocodazole, for the purpose of inducing aneuploidy. Next, 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the growth rate were investigated to select aneuploids. Aneuploids were selected at a frequency of 8.2 × 10−4, which was significantly higher than that of the control group, mainly at chromosomes I, II, III, IX, XII, XIII, and XVI. The acquired aneuploids were evaluated for their metabolic and brewing characteristics. A hierarchical cluster analysis based on endogenous metabolite data discriminated euploid S-3 and aneuploids. In addition, principal-component analysis of the constituents of the broth brewed with the strains discriminated between euploid S-3 and aneuploids. Sensory evaluation of the broth brewed with euploid S-3 and aneuploids showed that it tended to differ in aroma and taste. Specific ethanol production rates of the aneuploids were not deteriorated. The method of this selection made it possible to efficiently obtain aneuploids with various brewing characteristics from brewer's yeast, which do not correspond to genetically modified organisms. This novel breeding method focusing on chromosomal aneuploidy will facilitate the development of novel shochu yeast strains. [ABSTRACT FROM AUTHOR]

Alteration of the inward rectifier current I K 1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify I KIR 2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1-cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (* p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (* p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited I KIR 2.1. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes. [ABSTRACT FROM AUTHOR]

Alteration of the inward rectifier current IK1, carried by KIR2.1 channels, affects action potential duration, impacts resting membrane stability and associates with cardiac arrhythmias. Congenital and acquired KIR2.1 malfunction frequently associates with aberrant ion channel trafficking. Cellular processes underlying trafficking are intertwined with cytoskeletal function. The extent to which the cytoskeleton is involved in KIR2.1 trafficking processes is unknown. We aimed to quantify the dependence of KIR2.1 trafficking on cytoskeleton function. GFP or photoconvertible Dendra2 tagged KIR2.1 constructs were transfected in HEK293 or HeLa cells. Photoconversion of the Dendra2 probe at the plasma membrane and subsequent live imaging of trafficking processes was performed by confocal laser-scanning microscopy. Time constant of green fluorescent recovery (τg,s) represented recruitment of new KIR2.1 at the plasma membrane. Red fluorescent decay (τr,s) represented internalization of photoconverted KIR2.1. Patch clamp electrophysiology was used to quantify IKIR2.1. Biochemical methods were used for cytoskeleton isolation and detection of KIR2.1-cytoskeleton interactions. Cytochalasin B (20 μM), Nocodazole (30 μM) and Dyngo-4a (10 nM) were used to modify the cytoskeleton. Chloroquine (10 μM, 24 h) was used to impair KIR2.1 breakdown. Cytochalasin B and Nocodazole, inhibitors of actin and tubulin filament formation respectively, strongly inhibited the recovery of green fluorescence at the plasma membrane suggestive for inhibition of KIR2.1 forward trafficking [τg,s 13 ± 2 vs. 131 ± 31* and 160 ± 40* min, for control, Cytochalasin B and Nocodazole, respectively (*p < 0.05 vs. control)]. Dyngo-4a, an inhibitor of dynamin motor proteins, strongly slowed the rate of photoconverted channel internalization, whereas Nocodazole and Cytochalasin B had less effect [τr,s 20 ± 2 vs. 87 ± 14*, 60 ± 16 and 64 ± 20 min (*p < 0.05 vs. control)]. Cytochalasin B treatment (20 μM, 24 h) inhibited IKIR2.1. Chloroquine treatment (10 μM, 24 h) induced intracellular aggregation of KIR2.1 channels and enhanced interaction with the actin/intermediate filament system (103 ± 90 fold; p < 0.05 vs. control). Functional actin and tubulin cytoskeleton systems are essential for forward trafficking of KIR2.1 channels, whereas initial backward trafficking relies on a functional dynamin system. Chronic disturbance of the actin system inhibits KIR2.1 currents. Internalized KIR2.1 channels become recruited to the cytoskeleton, presumably in lysosomes.

The mechanical force between cells and the extracellular microenvironment is crucial to many physiological processes such as cancer metastasis and stem cell differentiation. Mitosis plays an essential role in all these processes and thus an in-depth understanding of forces during mitosis gains insight into disease diagnosis and disease treatment. Here, we develop a traction force microscope method based on monolayer fluorescent beads for measuring the weak traction force (tens of Pa) of mitotic cells in three dimensions. We quantify traction forces of human ovarian granulosa (KGN) cells exerted on the extracellular matrix throughout the entire cell cycle in three dimensions. Our measurements reveal how forces vary during the cell cycle, especially during cell division. Furthermore, we study the effect of paclitaxel (PTX) and nocodazole (NDZ) on mitotic KGN cells through the measurement of traction forces. Our results show that mitotic cells with high concentrations of PTX exert a larger force than those with high concentrations of NDZ, which proved to be caused by changes in the structure and number of microtubules. These findings reveal the key functions of microtubule in generating traction forces during cell mitosis and explain how dividing cells regulate themselves in response to anti-mitosis drugs. This work provides a powerful tool for investigating cell-matrix interactions during mitosis and may offer a potential way to new therapies for cancer. [ABSTRACT FROM AUTHOR]

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C(Cdc20) substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding "ABBA motif" in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C(Cdc20) substrate destruction.

Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

BackgroundCardiac dysfunction in failing hearts of human patients and animal models is associated with both microtubule densification and transverse-tubule (T-tubule) remodeling. Our objective was to investigate whether microtubule densification contributes to T-tubule remodeling and excitation-contraction coupling dysfunction in heart disease.Methods and resultsIn a mouse model of pressure overload-induced cardiomyopathy by transaortic banding, colchicine, a microtubule depolymerizer, significantly ameliorated T-tubule remodeling and cardiac dysfunction. In cultured cardiomyocytes, microtubule depolymerization with nocodazole or colchicine profoundly attenuated T-tubule impairment, whereas microtubule polymerization/stabilization with taxol accelerated T-tubule remodeling. In situ immunofluorescence of heart tissue sections demonstrated significant disorganization of junctophilin-2 (JP2), a protein that bridges the T-tubule and sarcoplasmic reticulum membranes, in transaortic banded hearts as well as in human failing hearts, whereas colchicine injection significantly preserved the distribution of JP2 in transaortic banded hearts. In isolated mouse cardiomyocytes, prolonged culture or treatment with taxol resulted in pronounced redistribution of JP2 from T-tubules to the peripheral plasma membrane, without changing total JP2 expression. Nocodazole treatment antagonized JP2 redistribution. Moreover, overexpression of a dominant-negative mutant of kinesin 1, a microtubule motor protein responsible for anterograde trafficking of proteins, protected against JP2 redistribution and T-tubule remodeling in culture. Finally, nocodazole treatment improved Ca(2+) handling in cultured myocytes by increasing the amplitude of Ca(2+) transients and reducing the frequency of Ca(2+) sparks.ConclusionOur data identify a mechanistic link between microtubule densification and T-tubule remodeling and reveal microtubule-mediated JP2 redistribution as a novel mechanism for T-tubule disruption, loss of excitation-contraction coupling, and heart failure.

The INK4A locus codes for two independent tumor suppressors, p14ARF and p16/CDKN2A, and is frequently mutated in many cancers. Here we report a novel deletion/substitution from CC to T in the shared exon 2 of p14ARF/p16 in a melanoma cell line. This mutation aligns the reading frames of p14ARF and p16 mid-transcript, producing one protein which is half p14ARF and half p16, chimera ARF (chARF), and another which is half p16 and half non-p14ARF/non-p16 amino acids, p16-Alternate Carboxyl Terminal (p16-ACT). In an effort to understand the cellular impact of this novel mutation and others like it, we expressed the two protein products in a tumor cell line and analyzed common p14ARF and p16 pathways, including the p53/p21 and CDK4/cyclin D1 pathways, as well as the influence of the two proteins on growth and the cell cycle. We report that chARF mimicked wild-type p14ARF by inducing the p53/p21 pathway, inhibiting cell growth through G2/M arrest and maintaining a certain percentage of cells in G1 during nocodazole-induced G2 arrest. chARF also demonstrated p16 activity by binding CDK4. However, rather than preventing cyclin D1 from binding CDK4, chARF stabilized this interaction through p21 which bound CDK4. p16-ACT had no p16-related function as it was unable to inhibit cyclin D1/CDK4 complex formation and was unable to arrest the cell cycle, though it did inhibit colony formation. We conclude that these novel chimeric proteins, which are very similar to predicted p16/p14ARF chimeric proteins found in other primary cancers, result in maintained p14ARF-p53-p21 signaling while p16-dependent CDK4 inhibition is lost.

Abstract Background Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

The intracellular protozoan parasite, Theileria annulata, induces uncontrolled proliferation and transformation in bovine B lymphocytes and monocytes in blood circulation andlymph nodes of host cells. This uncontrolled replication happens in the macroschizont stage of the life cycle of the parasites. The development of a rapid and efficient technique is likely to necessitate for isolation of purified schizonts from host cells. This is necessary for the isolation of highly purified RNA, protein or glycoproteins of schizonts from host cells. This study aimed to isolate the purified schizont based on the aerolysin–nocodazole technique. Aerolysin which was purified from gram-negative pathogen aeromonas hydrophila has an ability to form discrete channels and unstable eukaryotic cell membranes in 0C• and low concentration. Nocodazole used for parasite separation from the microtubule network of infected lymphocytes and monocytes. In purified schizonts, no large nucleus of host cells visualized in giemsa and DAPI staining. The isolated schizonts were free and intact from host cells. Intact T.annulata schizonts obtained from this study are suitable for purification of RNAs, proteins, glycolipids, and glycoproteins of schizonts free of host cell debris.

Abstract Background Drugs such as taxanes, epothilones, and vinca alkaloids are widely used in the treatment of breast, ovarian, and lung cancers but come with major side effects such as neuropathy and loss of neutrophils and as single agents have a lack of efficacy. M2I-1 (MAD2 inhibitor-1) has been shown to disrupt the CDC20-MAD2 interaction, and consequently, the assembly of the mitotic checkpoint complex (MCC). Results We report here that M2I-1 can significantly increase the sensitivity of several cancer cell lines to anti-mitotic drugs, with cell death occurring after a prolonged mitotic arrest. In the presence of nocodazole or taxol combined with M2I-1 cell death is triggered by the premature degradation of Cyclin B1, the perturbation of the microtubule network, and an increase in the level of the pro-apoptotic protein MCL-1s combined with a marginal increase in the level of NOXA. The elevated level of MCL-1s and the marginally increased NOXA antagonized the increased level of MCL-1, a pro-survival protein of the Bcl-2 family. Conclusion Our results provide some important molecular mechanisms for understanding the relationship between the mitotic checkpoint and programmed cell death and demonstrate that M2I-1 exhibits antitumor activity in the presence of current anti-mitotic drugs such as taxol and nocodazole and has the potential to be developed as an anticancer agent.

The adoption of new treatment modalities is required to improve outcomes in lung cancer treatment as lung cancer has lowest survival rates, along with liver and pancreatic cancer. Bortezomib is a proteasome inhibitor that has higher anticancer effect in combination therapies. The aim of this study was to investigate whether bortezomib could have additional anticancer effect with antineoplastic tubulin binding agent -nocodazole in A549 lung cancer cells. Apoptosis related gene expression levels of Noxa, Bcl-xL, Casp3 and Casp7 were measured by real-time PCR after treatment with 30 nM bortezomib, 0.3 µg/ml nocodazole and with their combination for 24 hours. Synergistic effect on DNA damage response was investigated at protein levels by checking p53 and cleaved PARP expressions. Induction of apoptotis was determined at protein expression level by western blotting of XIAP, Bcl-X and Bim. It was found that nocodazole combined bortezomib treatment induced apoptosis via p53 mediated DNA damage response signalling. P53 and cleaved PARP protein expressions were increased significantly after combination treatment. Apoptosis related genes Noxa, Casp3 and Casp7 mRNA expressions were elevated significantly after combination treatment. This study concludes that bortezomib potentiates the effect of nocodazole via DNA damage induced apoptosis in A549 lung cancer cells. [ABSTRACT FROM AUTHOR]

Multiciliate cells (MCCs) are highly specialized epithelial cells that employ hundreds of motile cilia to produce a vigorous directed flow in a variety of organ systems. The production of this flow requires the establishment of planar cell polarity (PCP) whereby MCCs align hundreds of beating cilia along a common planar axis. The planar axis of cilia in MCCs is known to be established via the PCP pathway and hydrodynamic cues, but the downstream steps required for cilia orientation remain poorly defined. Here, we describe a new component of cilia orientation, based on the phenotypic analysis of an uncharacterized coiled-coil protein, called bbof1. We show that the expression of bbof1 is induced during the early phases of MCC differentiation by the master regulator foxj1. MCC differentiation and ciliogenesis occurs normally in embryos where bbof1 activity is reduced, but cilia orientation is severely disrupted. We show that cilia in bbof1 mutants can still respond to patterning and hydrodynamic cues, but lack the ability to maintain their precise orientation. Misexpression of bbof1 promotes cilia alignment, even in the absence of flow or in embryos where microtubules and actin filaments are disrupted. Bbof1 appears to mediate cilia alignment by localizing to a polar structure adjacent to the basal body. Together, these results suggest that bbof1 is a basal body component required in MCCs to align and maintain cilia orientation in response to flow.

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.

BackgroundLipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors.ResultsIn this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome-DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes.ConclusionCollectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.

VEGF signaling is a key regulator of vessel formation and function. In vascular endothelial cells, this signaling is mediated through its cognate receptor VEGFR2, which is dynamically sorted in response to ligand. Little is known about the underlying mechanism of this intracellular sorting. Here we examined the role of different components of the cytoskeleton in this process. We found that VEGFR2 mainly associates with microtubule fibers and to a lesser extent with intermediate filaments and actin. Microtubule disruption leads to accumulation of VEGFR2 protein in the membrane and cytoplasm leading to defects in VEGF signaling. In contrast, inhibition of actin filaments results in no accumulation of VEGFR2 total protein or apparent changes in microtubule association. Instead, actin inhibition leads to a more global signaling disruption of the ERK1/2 pathway. This is the first report demonstrating that VEGFR2 associates closely with microtubules in modulating the subcellular sorting and signaling of VEGFR2.

Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We performed single-molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concentrations of H1 induce bending and looping of naked DNA at stretching forces below 0.6 pN, effects that can be reversed with 2.7-pN force or in 200 mM monovalent salt concentrations. Consecutive tens-of-nanometer bending events suggest that H1 binds to naked DNA in buffer at high stoichiometries. In egg extracts, single DNA molecules assemble into nucleosomes and undergo rapid compaction. Histone H1 at endogenous physiological concentrations increases the DNA compaction rate during chromatin assembly under 2-pN force and decreases it during disassembly under 5-pN force. In egg cytoplasm, histone H1 protects sperm nuclei undergoing genome-wide decondensation and chromatin assembly from becoming abnormally stretched or fragmented due to astral microtubule pulling forces. These results reveal functional ramifications of H1 binding to DNA at the single-molecule level and suggest an important physiological role for H1 in compacting DNA under force and during chromatin assembly.

Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.

The brain capillary endothelium is highly regulatory, maintaining the chemical stability of the brain’s microenvironment. The role of cytoskeletal proteins in tethering nanotubules (TENTs) during barrier-genesis was investigated using the established immortalized mouse brain endothelial cell line (bEnd5) as an in vitro blood-brain barrier (BBB) model. The morphology of bEnd5 cells was evaluated using both high-resolution scanning electron microscopy and immunofluorescence to evaluate treatment with depolymerizing agents Cytochalasin D for F-actin filaments and Nocodazole for α-tubulin microtubules. The effects of the depolymerizing agents were investigated on bEnd5 monolayer permeability by measuring the transendothelial electrical resistance (TEER). The data endorsed that during barrier-genesis, F-actin and α-tubulin play a cytoarchitectural role in providing both cell shape dynamics and cytoskeletal structure to TENTs forming across the paracellular space to provide cell-cell engagement. Western blot analysis of the treatments suggested a reduced expression of both proteins, coinciding with a reduction in the rates of cellular proliferation and decreased TEER. The findings endorsed that TENTs provide alignment of the paracellular (PC) spaces and tight junction (TJ) zones to occlude bEnd5 PC spaces. The identification of specific cytoskeletal structures in TENTs endorsed the postulate of their indispensable role in barrier-genesis and the maintenance of regulatory permeability across the BBB.

The first development method of brewery shochu yeast focusing on chromosomal aneuploidy is reported in this study. Euploidy diploid shochu yeast S-3 was treated with a microtubule inhibitor, nocodazole, for the purpose of inducing aneuploidy. Next, 2,3,5-triphenyl tetrazolium chloride (TTC) staining and the growth rate were investigated to select aneuploids. Aneuploids were selected at a frequency of 8.2 × 10−4, which was significantly higher than that of the control group, mainly at chromosomes I, II, III, IX, XII, XIII, and XVI. The acquired aneuploids were evaluated for their metabolic and brewing characteristics. A hierarchical cluster analysis based on endogenous metabolite data discriminated euploid S-3 and aneuploids. In addition, principal-component analysis of the constituents of the broth brewed with the strains discriminated between euploid S-3 and aneuploids. Sensory evaluation of the broth brewed with euploid S-3 and aneuploids showed that it tended to differ in aroma and taste. Specific ethanol production rates of the aneuploids were not deteriorated. The method of this selection made it possible to efficiently obtain aneuploids with various brewing characteristics from brewer’s yeast, which do not correspond to genetically modified organisms. This novel breeding method focusing on chromosomal aneuploidy will facilitate the development of novel shochu yeast strains.

The effect of microtubules disassembly with nocodazole and depolymerization of the actin cytoskeleton with cytochalasin D, as well as their combined effect on endocytosis dynamics of complexes of epidermal growth factor and its receptor in HeLa cells, was studied. Using confocal microscopy, it was shown that in cells fixed at different intervals after endocytosis stimulation, there is a high degree of colocalization of the receptor with the marker of early endosomes EEA1, which correlates with enlargement of endosomes as a result of fusion at early stages, and translocation of endosomes to the juxtanuclear area (JNA) at later stages. Translocation towards the JNA depends on microtubules. Nocodazole does not interfere with the formation of endosomes, but blocks their fusions and translocations. At the same time, depolymerization of actin microfilaments, not affecting the process of EEA1-dependent fusions and transport of endosomes into the JNA, leads to enlargement of endosomes, preventing the separation of early endosomes and mature multivesicular endosomes. With the destruction of both types of cytoskeleton, the newly formed receptor-containing endocytic vesicles do not enlarge, do not mature, and do not move into the JNA. Live cell imaging of endosomes with EGF associated with quantum dots confirmed the data obtained on fixed cells and made it possible to analyze the nature of an endosome motility. It turned out that rare periods of a fast linear run are changed for prolonged chaotic oscillations within a small area, while the directions of linear runs can be different, and not only towards the MTOC. Our data suggest that the role of microtubules is not only to ensure the translocations of endosomes, but also to provide a platform for their fusion. This assumption can also explain the seemingly chaotic movements of endosomes, which make it possible to increase the probability of their contacts and, consequently, fusions. [ABSTRACT FROM AUTHOR]

We conducted a study to decipher the mechanism of the formation of the large communal Leishmania amazonensis‐containing parasitophorous vacuole (PV) and found that the macrophage microtubule (MT) network dynamically orchestrates the intracellular lifestyle of this intracellular parasite. Physical disassembly of the MT network of macrophage‐like RAW 264.7 cells or silencing of the dynein gene, encoding the MT‐associated molecular motor that powers MT‐dependent vacuolar movement, by siRNA resulted in most of the infected cells hosting only tight parasite‐containing phagosome‐like vacuoles randomly distributed throughout the cytoplasm, each insulating a single parasite. Only a minority of the infected cells hosted both isolated parasite‐containing phagosome‐like vacuoles and a small communal PV, insulating a maximum of two to three parasites. The tight parasite‐containing phagosome‐like vacuoles never matured, whereas the small PVs only matured to a small degree, shown by the absence or faint acquisition of host‐cell endolysosomal characteristics. As a consequence, the parasites were unable to successfully complete promastigote‐to‐amastigote differentiation and died, regardless of the type of insulation. [ABSTRACT FROM AUTHOR]

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Pololike kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells. [ABSTRACT FROM AUTHOR]

In this study, a series of 2-Aryl-1 H -benzo[ d ]imidazole derivatives were developed to target intra- and extracellular microtubule networks. Compounds O-7 and O-10 showed impressive anti-proliferative activity across various tested cell lines, demonstrating selectivity indexes of 151.7 and 61.9, respectively. O-7 achieved an IC 50 value of 0.236 ± 0.096 μM, while O-10 showed an IC 50 value of 0.622 ± 0.13 μM against A549 cell lines. The induction of early-stage apoptosis in a dose-dependent manner further underscored the potential of O-7 and O-10 as effective anti-proliferative agents. O-7 and O-10 exhibited substantial inhibition of wound closure, with wound closure percentages decreasing from 23% at 0 μM to 0.43% and 2.62% at 20 μM, respectively. Colony formation reduction rates were impressive, with O-7 at 74.2% and O-10 at 81.2%. These results indicate that the O-7 and O-10 can impede cancer cell migration and have a high potential to curtail colony formation. The mode of action investigations for O-7 and O-10 revealed that O-7 could inhibit in vitro tubulin polymerization and disrupt the intracellular microtubule cytoskeleton. This disruption led to cell cycle arrest in the G 2 /M phase, indicating that O-7 exerts its anticancer activity through microtubule destabilization. However, O-10 shows a different mode of action than O-7 and requires further investigation. Overall, our study showcases the potential of the synthesized benzimidazole derivatives as novel and selective anticancer agents, motivating further exploration of their pharmacological properties and therapeutic applications. [Display omitted] Benzimidazole derivatives were designed to target the microtubule networks. Compounds O-7 and O-10 showed selectivity indexes of 151.7 and 61.9, respectively. O-7 (IC 50 = 0.236) and O-10 (IC 50 = 0.622) showed potent anticancer activity. O-7 impede cancer cell migration by inhibiting wound closure and colony formation. MoA of O-7 indicates G 2/M cell cycle arrest followed by early appoptosis. [ABSTRACT FROM AUTHOR]

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes' motion.

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes' motion.

In eukaryotic cells, cohesin holds sister chromatids together until they separate into daughter cells during mitosis. We have used chromatin immunoprecipitation coupled with microarray analysis (ChIP chip) to produce a genome-wide description of cohesin binding to meiotic and mitotic chromosomes of Saccharomyces cerevisiae. A computer program, PeakFinder, enables flexible, automated identification and annotation of cohesin binding peaks in ChIP chip data. Cohesin sites are highly conserved in meiosis and mitosis, suggesting that chromosomes share a common underlying structure during different developmental programs. These sites occur with a semiperiodic spacing of 11 kb that correlates with AT content. The number of sites correlates with chromosome size; however, binding to neighboring sites does not appear to be cooperative. We observed a very strong correlation between cohesin sites and regions between convergent transcription units. The apparent incompatibility between transcription and cohesin binding exists in both meiosis and mitosis. Further experiments reveal that transcript elongation into a cohesin-binding site removes cohesin. A negative correlation between cohesin sites and meiotic recombination sites suggests meiotic exchange is sensitive to the chromosome structure provided by cohesin. The genome-wide view of mitotic and meiotic cohesin binding provides an important framework for the exploration of cohesins and cohesion in other genomes.

Nocodazole is an antineoplastic agent that exerts its effects by depolymerizing microtubules. Herein we report a structural analog of nocodazole, a (1H-pyrrol-1-yl)methyl-1H-benzoimidazole carbamate ester derivative, named RDS 60. We evaluated the antineoplastic properties of RDS 60 in two human head and neck squamous cell carcinoma (HNSCC) cell lines and we found that this compound significantly inhibited replication of both HNSCC cell lines without inducing any important cytotoxic effect on human dermal fibroblasts and human keratinocytes. The treatment of HNSCC cell lines with 1 μM RDS 60 for 24 h stopped development of normal bipolar mitotic spindles and, at the same time, blocked the cell cycle in G2/M phase together with cytoplasmic accumulation of cyclin B1. Consequently, treatment with 2 μM RDS 60 for 24 h induced the activation of apoptosis in both HNSCC cell lines. Additionally, RDS 60 was able to reverse the epithelial-mesenchymal transition and to inhibit cell migration and extracellular matrix infiltration of both HNSCC cell lines. The reported results demonstrate that this compound has a potent effect in blocking cell cycle, inducing apoptosis and inhibiting cell motility and stromal invasion of HNSCC cell lines. Therefore, the ability of RDS 60 to attenuate the malignancy of tumor cells suggests its potential role as an interesting and powerful tool for new approaches in treating HNSCC.

Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors in vitro and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While the mechanisms of MT inhibitors’ interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated “trigger” concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the “trigger” promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

A series of new amide derivatives of imidazopyridine is synthesized and structures of the products are confirmed by 1H and 13C NMR, and mass spectral data. The synthesized derivatives are screened for their anticancer activity against four human cancer cell lines: lung cancer (A549), breast cancer (MCF-7), melanoma cancer (A375), and colon cancer (HT-29). Six synthesized compounds exhibit more potent activity than the control drug. [ABSTRACT FROM AUTHOR]