Alisa Piekny - Academia.edu (original) (raw)
Papers by Alisa Piekny
Journal of cell science, 2021
Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. M... more Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Fina...
Bioorganic & Medicinal Chemistry Letters, 2021
Several derivatives of a series that share a thienoisoquinoline scaffold have demonstrated potent... more Several derivatives of a series that share a thienoisoquinoline scaffold have demonstrated potent anti-cancer activity against cancer cell lines A549, HeLa, HCT-116 in the submicromolar concentration range. Structure-activity relationship (SAR) studies on a range of derivatives aided in identifying key pharmacophores in the lead compound. A series of compounds have been identified as the most promising with submicromolar IC50 values against a lung cancer cell line (A549). Microscopy studies of cancer cells treated with the lead compound revealed that it causes mitotic arrest and disrupts microtubules. Further evaluation via an in vitro microtubule polymerization assay and competition studies indicate that the lead compound binds to tubulin via the colchicine site.
Developmental Biology, 2013
The formation of tissues is essential for metazoan development. During Caenorhabditis elegans emb... more The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues.
Frontiers in Cell and Developmental Biology
Cytokinesis is required to physically cleave a cell into two daughters at the end of mitosis. Dec... more Cytokinesis is required to physically cleave a cell into two daughters at the end of mitosis. Decades of research have led to a comprehensive understanding of the core cytokinesis machinery and how it is regulated in animal cells, however this knowledge was generated using single cells cultured in vitro, or in early embryos before tissues develop. This raises the question of how cytokinesis is regulated in diverse animal cell types and developmental contexts. Recent studies of distinct cell types in the same organism or in similar cell types from different organisms have revealed striking differences in how cytokinesis is regulated, which includes different threshold requirements for the structural components and the mechanisms that regulate them. In this review, we highlight these differences with an emphasis on pathways that are independent of the mitotic spindle, and operate through signals associated with the cortex, kinetochores, or chromatin.
Scientific Reports
We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core ... more We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymeriz...
Applied and Environmental Microbiology, 2022
One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant... more One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant produced by a range of unrelated microbes. Many animals, including humans, are able to detect geosmin at minute concentrations, but the benefit that this compound provides to its producing organisms is poorly understood.
Poster 1734C for TAGC 2020 Online. File is a PDF of the poster (TAGC virtual conference.PDF)
Journal of Cell Science, 2022
Cytokinesis occurs at the end of mitosis due to the ingression of a contractile ring that cleaves... more Cytokinesis occurs at the end of mitosis due to the ingression of a contractile ring that cleaves the daughter cells. The core machinery regulating this crucial process is conserved among metazoans. Multiple pathways control ring assembly, but their contribution in different cell types is not known. We found that in the C. elegans embryo, AB and P1 cells fated to be somatic tissue and germline, respectively, have different cytokinesis kinetics supported by distinct myosin levels and organization. Through perturbation of RhoA or polarity regulators and the generation of tetraploid strains, we found that ring assembly is controlled by multiple fate-dependent factors that include myosin-levels, and mechanisms that respond to cell size. Active Ran coordinates ring position with the segregating chromatids in HeLa cells by forming an inverse gradient with importins that control the cortical recruitment of anillin. We found that the Ran pathway regulates anillin in AB cells, but functions ...
In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression ... more In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the central spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the central spindle by binding to centralspindlin. Depletion of the centralspindlin component
Proceedings of the 3rd International Conference on Theoretical and Applied Nanoscience and Nanotechnology (TANN'19), 2019
There is a growing demand for non-toxic metallic nanoparticles for biomedical imaging, targeted d... more There is a growing demand for non-toxic metallic nanoparticles for biomedical imaging, targeted drug delivery and other therapeutic applications. Metallic nanoparticle based cancer therapy could be superior to conventional chemotherapy, where targeted drug delivery is required. Incinerated ash (parpam) of Gold (Au) (Swarna parpam), Silver (Ag) (Velli parpam), Copper (Cu) (Sembu parpam, and Iron (Fe) (Aya parpam), have been used for the past many100 years, by Siddha, Ayurveda and Unani, the three traditional medical systems in India. They are used to treat various chronic diseases, including cancer. The respective metals are treated with herbal extracts and incinerated into nano/micro particles, suitable for oral administration. Following this, our group reported the effect of incinerated Au particles on normal and cancerous cells, their behavior inside these cells-nuclear accumulation, cell entry pathways and toxicity1. Our work is the initial step towards using metallic particles used in the Indian medical system for targeted cancer therapy. We reported that the Indian Au nano particles were found non-toxic and localized in the different parts of the HeLa cells through our examinations using hyperspectral microscopy, SEM and Raman spectroscopy. As a next step, we plan to examine Ag nanoparticles interaction with cancer cells. The specificity in their effects on respective cancer cells, cervical cancer (HeLa), Colon cancer (HCT 116) and lung cancer (A549), needs to be elucidated. This will aid in understanding the behavior of different nano-metals inside the cancer cells. Objective: Investigate incinerated metal nano/micro particles (Ag, Fe and C) used in Indian medicine to treat cancer. Novelty: The intracellular behavior of Ag nanoparticles, used in the Indian medicine and their effect on different cancer cells are studied for the first time. Method: The cellular intake of the nanoparticles are studied through cellular entry assays and hyperspectral imaging. The protein profiles will be determined through immunochemistry studies, respectively. The physiochemical properties of the nanoparticles will be characterized using, SEM, EDS and DLS. Results: Hyperspectral imaging was used to analyses these metal ashes (parpams) for the first time. The size of the metal ashes range from a few nm to <1000 nm. The preliminary results shows that the particles did not have significant effect on the cell proliferation of HeLa, HCT 116 and A549. The Indian Ag particles were found to enter into the A549 cells. Scope: This study will lead to the use of specific metal nanoparticles for particular cancer conditions. Further, this work will open the door to novel metallic particles for multidirectional biomedical applications. Conclusion: Nanomedicine is considered as the last option when treating cancer and other chronic diseases by the Indian medical system. But the pharmacokinetics of these drugs are not been elucidated. Our work will aid in understanding the effect of these drugs at the cellular level, resulting in the safe use of them at the early stages of cancer.
Small GTPases, 2021
Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spat... more Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.
Proceedings of the 2nd International Conference of Theoretical and Applied Nanoscience and Nanotechnology (TANN'18), 2018
ABSTRACTCytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractil... more ABSTRACTCytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent cell fate changes or aneuploidy, and the core machinery is highly conserved among metazoans. Multiple mechanisms regulate cytokinesis, but their requirement in different cell types is not known. Here, we show that differently fated AB and P1 cells in the early C. elegans embryo have unique cytokinesis kinetics supported by distinct levels and cortical patterning of myosin. Through perturbation of polarity regulators and the generation of stable tetraploid strains, we demonstrate that these differences depend on both cell fate and size. Additionally, these parameters could influence the Ran pathway, which coordinates the contractile ring with chromatin position, and controls cytokinesis differently in AB and P1 cells. Our findings demonstrate the need to consider multiple parameters when modeling ring ki...
Proceedings of the 4th World Congress on Recent Advances in Nanotechnology, Apr 1, 2019
Despite many advancements in detection and treatment, cancer remains the main cause of death in t... more Despite many advancements in detection and treatment, cancer remains the main cause of death in the world. During the cancer progression, the functional behaviour of cells such as adhesion, invasion, migration, and signalling are altered [1]. These changes can be accompanied by the alteration of the biophysical properties of cells. It has been substantiated that there is a correlation between the metastasis potential of cells and their mechanobiological properties. The stiffness of cancer cells is reduced compared to their healthy counterparts, enabling them to pass through endothelial cells, enter into the circulation system and invade the neighbouring tissues. This process, the metastasis, is the main reason for the deaths due to cancer [2]. The mechanical properties of cells are determined by the cytoskeletal structure and during the cancer progression, this structure is reorganized, enabling cancer cells to change from immotile state to motile state[3][4]. Nanotechnology has provided a great platform to develop nano-tools for detection and treatment of cancer. With the help of nanotechnology, nanoparticles can be designed to target the cytoskeletal proteins to regulate the metastasis level of cancer cells. Direct or indirect interaction of nanoparticles with filamentous proteins affects the structural remodelling of cancer cells and ultimately can hamper or reduce the dynamical capacity of cells. Due to the disturbing influence of nanoparticles, the migration and motility ability of cells can be changed[5][6]. In the current work, we have studied the interaction of two different gold nanoparticles (nanosphere and nanostar-shaped) with lung human cancer cells (A549) to investigate their cytotoxicity as well as their impacts on the cellular mechanobiology. The two types of Au nanoparticles were synthesised by chemical methods [7] [8] by reduction of a gold precursor and characterized with different techniques: Scanning Electron Microscopy (SEM), UV/Visible spectroscopy and Light Dynamic Scattering (DLS). SEM and DLS measurements showed that the average diameter of nanospheres and nanostars are ~22 nm and ~80 nm respectively. Gold nanoparticles appeared to be non-toxic to the cells as no cell death was observed; however, some disruptions have been seen in the cytoskeletal structure of cells. Atomic Force Microscopy (AFM) and scratch assays were utilized to characterize the stiffness and the migration ability of cells upon interaction with the gold nanoparticles. The results showed that the stiffness and migration ability of A549 cells are influenced by nanoparticles. However, the level of the effect depends on the shape and size of nanoparticles. Our results showed that the stiffness of cells is reduced by 50% after interaction with gold nanoparticles, confirming their potential to significantly change the cellular mechanobiology. The results showed that star-shaped particles are less effective in changing the mechanobiological properties of cancer cells, likely due to their shape and size. These preliminary data illustrate the potential applications of gold nanoparticles in inhibiting cancer metastasis.
The EMBO Journal, 2008
Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytos... more Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.
Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at pic... more Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis we found that geosmin and the related terpene 2-methylisoborneol reduced predation of Streptomyces coelicolor and Myxococcus xanthus by the bacteriophagous Caenorhabditis elegans. Predation was restored by the removal of both terpene biosynthetic pathways or deletion of the C. elegans ASE sensory neuron, and resulted in the death of the nematodes. Geosmin itself was non-toxic. This is the first warning chemical to be identified in bacteria or fungi, and suggests molecular signalling affects microbial predator-prey interactions in a...
Microsystems & Nanoengineering
Determining the characteristics and localization of nanoparticles inside cells is crucial for nan... more Determining the characteristics and localization of nanoparticles inside cells is crucial for nanomedicine design for cancer therapy. Hyperspectral imaging is a fast, straightforward, reliable, and accurate method to study the interactions of nanoparticles and intracellular components. With a hyperspectral image, we could collect spectral information consisting of thousands of pixels in a short time. Using hyperspectral images, in this work, we developed a label-free technique to detect nanoparticles in different regions of the cell. This technique is based on plasmonic shifts taking place during the interaction of nanoparticles with the surrounding medium. The unique optical properties of gold nanoparticles, localized surface plasmon resonance bands, are influenced by their microenvironment. The LSPR properties of nanoparticles, hence, could provide information on regions in which nanoparticles are distributed. To examine the potential of this technique for intracellular detection,...
International Journal of Theoretical and Applied Nanotechnology
ABSTRACTWe characterize the mechanism of action of a new microtubule-targeting compound in cells.... more ABSTRACTWe characterize the mechanism of action of a new microtubule-targeting compound in cells. Microtubule-targeting drugs are used as successful anti-cancer therapies. We synthesized a family of compounds that share a common scaffold and have several functional groups amenable to modifications. We found that one of the active derivatives, C75, reduces cell viability and prevents microtubule polymerization in vitro. In this study, we explore the phenotypes caused by C75 in cells. It causes mitotic arrest and spindle phenotypes in several cancer cell lines in the nanomolar range. C75 can bind to the Colchicine-pocket on tubulin in vitro, but causes different effects on microtubules in cells. While Colchicine causes a decrease in microtubules and spindle pole collapse without re-growth, similar concentrations of C75 cause a rapid loss of microtubules and spindle pole fragmentation followed by microtubule re-growth to form multipolar spindles. In addition, C75 and Colchicine synergi...
Journal of cell science, 2021
Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. M... more Cytokinesis is the process that separates a cell into two daughter cells at the end of mitosis. Most of our knowledge of cytokinesis comes from overexpression studies, which affects our interpretation of protein function. Gene editing can circumvent this issue by introducing functional mutations or fluorescent probes directly into a gene locus. However, despite its potential, gene editing is just starting to be used in the field of cytokinesis. Here, we discuss the benefits of using gene editing tools for the study of cytokinesis and highlight recent studies that successfully used CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) technology to answer critical questions regarding the function of cytokinesis proteins. We also present methodologies for editing essential genes and discuss how CRISPR interference (CRISPRi) and activation (CRISPRa) can enable precise control of gene expression to answer important questions in the field. Fina...
Bioorganic & Medicinal Chemistry Letters, 2021
Several derivatives of a series that share a thienoisoquinoline scaffold have demonstrated potent... more Several derivatives of a series that share a thienoisoquinoline scaffold have demonstrated potent anti-cancer activity against cancer cell lines A549, HeLa, HCT-116 in the submicromolar concentration range. Structure-activity relationship (SAR) studies on a range of derivatives aided in identifying key pharmacophores in the lead compound. A series of compounds have been identified as the most promising with submicromolar IC50 values against a lung cancer cell line (A549). Microscopy studies of cancer cells treated with the lead compound revealed that it causes mitotic arrest and disrupts microtubules. Further evaluation via an in vitro microtubule polymerization assay and competition studies indicate that the lead compound binds to tubulin via the colchicine site.
Developmental Biology, 2013
The formation of tissues is essential for metazoan development. During Caenorhabditis elegans emb... more The formation of tissues is essential for metazoan development. During Caenorhabditis elegans embryogenesis, ventral epidermal cells migrate to encase the ventral surface of the embryo in a layer of epidermis by a process known as ventral enclosure. This process is regulated by guidance cues secreted by the underlying neuroblasts. However, since the cues and their receptors are differentially expressed in multiple cell types, the role of the neuroblasts in ventral enclosure is not fully understood. Furthermore, although F-actin is required for epidermal cell migration, it is not known if nonmuscle myosin is also required. Anillin (ANI-1) is an actin and myosin-binding protein that coordinates actin-myosin contractility in the early embryo. Here, we show that ANI-1 localizes to the cleavage furrows of dividing neuroblasts during mid-embryogenesis and is required for their division. Embryos depleted of ani-1 display a range of ventral enclosure phenotypes, where ventral epidermal cells migrate with similar speeds to control embryos, but contralateral neighbors often fail to meet and are misaligned. The ventral enclosure phenotypes in ani-1 RNAi embryos suggest that the position or shape of neuroblasts is important for directing ventral epidermal cell migration, although does not rule out an autonomous requirement for ani-1 in the epidermal cells. Furthermore, we show that rho-1 and other regulators of nonmuscle myosin activity are required for ventral epidermal cell migration. Interestingly, altering nonmuscle myosin contractility alleviates or strengthens ani-1's ventral enclosure phenotypes. Our findings suggest that ventral enclosure is a complex process that likely relies on inputs from multiple tissues.
Frontiers in Cell and Developmental Biology
Cytokinesis is required to physically cleave a cell into two daughters at the end of mitosis. Dec... more Cytokinesis is required to physically cleave a cell into two daughters at the end of mitosis. Decades of research have led to a comprehensive understanding of the core cytokinesis machinery and how it is regulated in animal cells, however this knowledge was generated using single cells cultured in vitro, or in early embryos before tissues develop. This raises the question of how cytokinesis is regulated in diverse animal cell types and developmental contexts. Recent studies of distinct cell types in the same organism or in similar cell types from different organisms have revealed striking differences in how cytokinesis is regulated, which includes different threshold requirements for the structural components and the mechanisms that regulate them. In this review, we highlight these differences with an emphasis on pathways that are independent of the mitotic spindle, and operate through signals associated with the cortex, kinetochores, or chromatin.
Scientific Reports
We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core ... more We reveal the effects of a new microtubule-destabilizing compound in human cells. C75 has a core thienoisoquinoline scaffold with several functional groups amenable to modification. Previously we found that sub micromolar concentrations of C75 caused cytotoxicity. We also found that C75 inhibited microtubule polymerization and competed with colchicine for tubulin-binding in vitro. However, here we found that the two compounds synergized suggesting differences in their mechanism of action. Indeed, live imaging revealed that C75 causes different spindle phenotypes compared to colchicine. Spindles remained bipolar and collapsed after colchicine treatment, while C75 caused bipolar spindles to become multipolar. Importantly, microtubules rapidly disappeared after C75-treatment, but then grew back unevenly and from multiple poles. The C75 spindle phenotype is reminiscent of phenotypes caused by depletion of ch-TOG, a microtubule polymerase, suggesting that C75 blocks microtubule polymeriz...
Applied and Environmental Microbiology, 2022
One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant... more One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant produced by a range of unrelated microbes. Many animals, including humans, are able to detect geosmin at minute concentrations, but the benefit that this compound provides to its producing organisms is poorly understood.
Poster 1734C for TAGC 2020 Online. File is a PDF of the poster (TAGC virtual conference.PDF)
Journal of Cell Science, 2022
Cytokinesis occurs at the end of mitosis due to the ingression of a contractile ring that cleaves... more Cytokinesis occurs at the end of mitosis due to the ingression of a contractile ring that cleaves the daughter cells. The core machinery regulating this crucial process is conserved among metazoans. Multiple pathways control ring assembly, but their contribution in different cell types is not known. We found that in the C. elegans embryo, AB and P1 cells fated to be somatic tissue and germline, respectively, have different cytokinesis kinetics supported by distinct myosin levels and organization. Through perturbation of RhoA or polarity regulators and the generation of tetraploid strains, we found that ring assembly is controlled by multiple fate-dependent factors that include myosin-levels, and mechanisms that respond to cell size. Active Ran coordinates ring position with the segregating chromatids in HeLa cells by forming an inverse gradient with importins that control the cortical recruitment of anillin. We found that the Ran pathway regulates anillin in AB cells, but functions ...
In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression ... more In anaphase, the spindle dictates the site of contractile ring assembly. Assembly and ingression of the contractile ring involves activation of myosin-II and actin polymerization, which are triggered by the GTPase RhoA. In many cells, the central spindle affects division plane positioning via unknown molecular mechanisms. Here, we dissect furrow formation in human cells and show that the RhoGEF ECT2 is required for cortical localization of RhoA and contractile ring assembly. ECT2 concentrates on the central spindle by binding to centralspindlin. Depletion of the centralspindlin component
Proceedings of the 3rd International Conference on Theoretical and Applied Nanoscience and Nanotechnology (TANN'19), 2019
There is a growing demand for non-toxic metallic nanoparticles for biomedical imaging, targeted d... more There is a growing demand for non-toxic metallic nanoparticles for biomedical imaging, targeted drug delivery and other therapeutic applications. Metallic nanoparticle based cancer therapy could be superior to conventional chemotherapy, where targeted drug delivery is required. Incinerated ash (parpam) of Gold (Au) (Swarna parpam), Silver (Ag) (Velli parpam), Copper (Cu) (Sembu parpam, and Iron (Fe) (Aya parpam), have been used for the past many100 years, by Siddha, Ayurveda and Unani, the three traditional medical systems in India. They are used to treat various chronic diseases, including cancer. The respective metals are treated with herbal extracts and incinerated into nano/micro particles, suitable for oral administration. Following this, our group reported the effect of incinerated Au particles on normal and cancerous cells, their behavior inside these cells-nuclear accumulation, cell entry pathways and toxicity1. Our work is the initial step towards using metallic particles used in the Indian medical system for targeted cancer therapy. We reported that the Indian Au nano particles were found non-toxic and localized in the different parts of the HeLa cells through our examinations using hyperspectral microscopy, SEM and Raman spectroscopy. As a next step, we plan to examine Ag nanoparticles interaction with cancer cells. The specificity in their effects on respective cancer cells, cervical cancer (HeLa), Colon cancer (HCT 116) and lung cancer (A549), needs to be elucidated. This will aid in understanding the behavior of different nano-metals inside the cancer cells. Objective: Investigate incinerated metal nano/micro particles (Ag, Fe and C) used in Indian medicine to treat cancer. Novelty: The intracellular behavior of Ag nanoparticles, used in the Indian medicine and their effect on different cancer cells are studied for the first time. Method: The cellular intake of the nanoparticles are studied through cellular entry assays and hyperspectral imaging. The protein profiles will be determined through immunochemistry studies, respectively. The physiochemical properties of the nanoparticles will be characterized using, SEM, EDS and DLS. Results: Hyperspectral imaging was used to analyses these metal ashes (parpams) for the first time. The size of the metal ashes range from a few nm to <1000 nm. The preliminary results shows that the particles did not have significant effect on the cell proliferation of HeLa, HCT 116 and A549. The Indian Ag particles were found to enter into the A549 cells. Scope: This study will lead to the use of specific metal nanoparticles for particular cancer conditions. Further, this work will open the door to novel metallic particles for multidirectional biomedical applications. Conclusion: Nanomedicine is considered as the last option when treating cancer and other chronic diseases by the Indian medical system. But the pharmacokinetics of these drugs are not been elucidated. Our work will aid in understanding the effect of these drugs at the cellular level, resulting in the safe use of them at the early stages of cancer.
Small GTPases, 2021
Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spat... more Cytokinesis is required to cleave the daughter cells at the end of mitosis and relies on the spatiotemporal control of RhoA GTPase. Cytokinesis failure can lead to changes in cell fate or aneuploidy, which can be detrimental during development and/or can lead to cancer. However, our knowledge of the pathways that regulate RhoA during cytokinesis is limited, and the role of other Rho family GTPases is not clear. This is largely because the study of Rho GTPases presents unique challenges using traditional cell biological and biochemical methods, and they have pleiotropic functions making genetic studies difficult to interpret. The recent generation of optogenetic tools and biosensors that control and detect active Rho has overcome some of these challenges and is helping to elucidate the role of RhoA in cytokinesis. However, improvements are needed to reveal the role of other Rho GTPases in cytokinesis, and to identify the molecular mechanisms that control Rho activity. This review examines some of the outstanding questions in cytokinesis, and explores tools for the imaging and control of Rho GTPases.
Proceedings of the 2nd International Conference of Theoretical and Applied Nanoscience and Nanotechnology (TANN'18), 2018
ABSTRACTCytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractil... more ABSTRACTCytokinesis occurs at the end of mitosis and occurs due to the ingression of a contractile ring that cleaves the daughter cells. This process is tightly controlled to prevent cell fate changes or aneuploidy, and the core machinery is highly conserved among metazoans. Multiple mechanisms regulate cytokinesis, but their requirement in different cell types is not known. Here, we show that differently fated AB and P1 cells in the early C. elegans embryo have unique cytokinesis kinetics supported by distinct levels and cortical patterning of myosin. Through perturbation of polarity regulators and the generation of stable tetraploid strains, we demonstrate that these differences depend on both cell fate and size. Additionally, these parameters could influence the Ran pathway, which coordinates the contractile ring with chromatin position, and controls cytokinesis differently in AB and P1 cells. Our findings demonstrate the need to consider multiple parameters when modeling ring ki...
Proceedings of the 4th World Congress on Recent Advances in Nanotechnology, Apr 1, 2019
Despite many advancements in detection and treatment, cancer remains the main cause of death in t... more Despite many advancements in detection and treatment, cancer remains the main cause of death in the world. During the cancer progression, the functional behaviour of cells such as adhesion, invasion, migration, and signalling are altered [1]. These changes can be accompanied by the alteration of the biophysical properties of cells. It has been substantiated that there is a correlation between the metastasis potential of cells and their mechanobiological properties. The stiffness of cancer cells is reduced compared to their healthy counterparts, enabling them to pass through endothelial cells, enter into the circulation system and invade the neighbouring tissues. This process, the metastasis, is the main reason for the deaths due to cancer [2]. The mechanical properties of cells are determined by the cytoskeletal structure and during the cancer progression, this structure is reorganized, enabling cancer cells to change from immotile state to motile state[3][4]. Nanotechnology has provided a great platform to develop nano-tools for detection and treatment of cancer. With the help of nanotechnology, nanoparticles can be designed to target the cytoskeletal proteins to regulate the metastasis level of cancer cells. Direct or indirect interaction of nanoparticles with filamentous proteins affects the structural remodelling of cancer cells and ultimately can hamper or reduce the dynamical capacity of cells. Due to the disturbing influence of nanoparticles, the migration and motility ability of cells can be changed[5][6]. In the current work, we have studied the interaction of two different gold nanoparticles (nanosphere and nanostar-shaped) with lung human cancer cells (A549) to investigate their cytotoxicity as well as their impacts on the cellular mechanobiology. The two types of Au nanoparticles were synthesised by chemical methods [7] [8] by reduction of a gold precursor and characterized with different techniques: Scanning Electron Microscopy (SEM), UV/Visible spectroscopy and Light Dynamic Scattering (DLS). SEM and DLS measurements showed that the average diameter of nanospheres and nanostars are ~22 nm and ~80 nm respectively. Gold nanoparticles appeared to be non-toxic to the cells as no cell death was observed; however, some disruptions have been seen in the cytoskeletal structure of cells. Atomic Force Microscopy (AFM) and scratch assays were utilized to characterize the stiffness and the migration ability of cells upon interaction with the gold nanoparticles. The results showed that the stiffness and migration ability of A549 cells are influenced by nanoparticles. However, the level of the effect depends on the shape and size of nanoparticles. Our results showed that the stiffness of cells is reduced by 50% after interaction with gold nanoparticles, confirming their potential to significantly change the cellular mechanobiology. The results showed that star-shaped particles are less effective in changing the mechanobiological properties of cancer cells, likely due to their shape and size. These preliminary data illustrate the potential applications of gold nanoparticles in inhibiting cancer metastasis.
The EMBO Journal, 2008
Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytos... more Cytokinesis is a highly regulated and dynamic event that involves the reorganization of the cytoskeleton and membrane compartments. Recently, FIP3 has been implicated in targeting of recycling endosomes to the mid-body of dividing cells and is found required for abscission. Here, we demonstrate that the centralspindlin component Cyk-4 is a FIP3-binding protein. Furthermore, we show that FIP3 binds to Cyk-4 at late telophase and that centralspindlin may be required for FIP3 recruitment to the mid-body. We have mapped the FIP3-binding region on Cyk-4 and show that it overlaps with the ECT2-binding domain. Finally, we demonstrate that FIP3 and ECT2 form mutually exclusive complexes with Cyk-4 and that dissociation of ECT2 from the mid-body at late telophase may be required for the recruitment of FIP3 and recycling endosomes to the cleavage furrow. Thus, we propose that centralspindlin complex not only regulates acto-myosin ring contraction but also endocytic vesicle transport to the cleavage furrow and it does so through sequential interactions with ECT2 and FIP3.
Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at pic... more Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis we found that geosmin and the related terpene 2-methylisoborneol reduced predation of Streptomyces coelicolor and Myxococcus xanthus by the bacteriophagous Caenorhabditis elegans. Predation was restored by the removal of both terpene biosynthetic pathways or deletion of the C. elegans ASE sensory neuron, and resulted in the death of the nematodes. Geosmin itself was non-toxic. This is the first warning chemical to be identified in bacteria or fungi, and suggests molecular signalling affects microbial predator-prey interactions in a...
Microsystems & Nanoengineering
Determining the characteristics and localization of nanoparticles inside cells is crucial for nan... more Determining the characteristics and localization of nanoparticles inside cells is crucial for nanomedicine design for cancer therapy. Hyperspectral imaging is a fast, straightforward, reliable, and accurate method to study the interactions of nanoparticles and intracellular components. With a hyperspectral image, we could collect spectral information consisting of thousands of pixels in a short time. Using hyperspectral images, in this work, we developed a label-free technique to detect nanoparticles in different regions of the cell. This technique is based on plasmonic shifts taking place during the interaction of nanoparticles with the surrounding medium. The unique optical properties of gold nanoparticles, localized surface plasmon resonance bands, are influenced by their microenvironment. The LSPR properties of nanoparticles, hence, could provide information on regions in which nanoparticles are distributed. To examine the potential of this technique for intracellular detection,...
International Journal of Theoretical and Applied Nanotechnology
ABSTRACTWe characterize the mechanism of action of a new microtubule-targeting compound in cells.... more ABSTRACTWe characterize the mechanism of action of a new microtubule-targeting compound in cells. Microtubule-targeting drugs are used as successful anti-cancer therapies. We synthesized a family of compounds that share a common scaffold and have several functional groups amenable to modifications. We found that one of the active derivatives, C75, reduces cell viability and prevents microtubule polymerization in vitro. In this study, we explore the phenotypes caused by C75 in cells. It causes mitotic arrest and spindle phenotypes in several cancer cell lines in the nanomolar range. C75 can bind to the Colchicine-pocket on tubulin in vitro, but causes different effects on microtubules in cells. While Colchicine causes a decrease in microtubules and spindle pole collapse without re-growth, similar concentrations of C75 cause a rapid loss of microtubules and spindle pole fragmentation followed by microtubule re-growth to form multipolar spindles. In addition, C75 and Colchicine synergi...