quantitative-immunofluorescence-assay-to-measure-variation-protein

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

quantitative-pcr-based-assay-to-measure-sonic-hedgehog-signaling

Quantitative PCR-based Assay to Measure Sonic Hedgehog Signaling in Cellular Model of Ciliogenesis

Ciliopathy refers to a collection of multisystemic and polygenic human diseases and disorders that arise due to aberration of structure or function of ...

The Ohio State University

Presidency University

Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1x to 6x or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8-12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20-25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1x and 2x unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors - monastrol and HR22C16 analogues.

Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica.

Clostridial glucosylating cytotoxins inactivate mammalian Rho GTPases by mono-O glucosylation of a conserved threonine residue located in the switch 1 region of the target protein. Here we report that EhRho1, a RhoA-like GTPase from the protozoan parasite Entamoeba histolytica, is glucosylated by clostridial cytotoxins. Recombinant glutathione S-transferase-EhRho1 and EhRho1 from cell lysate of Entamoeba histolytica were glucosylated by Clostridium difficile toxin B and Clostridium novyi alpha-toxin. In contrast, Clostridium difficile toxin A, which shares the same mammalian protein substrates with toxin B, did not modify EhRho1. Change of threonine 52 of EhRho1 to alanine prevented glucosylation by toxin B from Clostridium difficile and by alpha-toxin from Clostridium novyi, which suggests that the equivalent threonine residues are glucosylated in mammalian and Entamoeba Rho GTPases. Lethal toxin from Clostridium sordellii did not glucosylate EhRho1 but labeled several other substrate proteins in lysates from Entamoeba histolytica in the presence of UDP-[14C]glucose.

EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins.

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.

Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica.

The formin family of proteins mediates dynamic changes in actin assembly in eukaryotes, and therefore it is important to understand the function of these proteins in Entamoeba histolytica, where actin forms the major cytoskeletal network. In this study we have identified the formin homologs encoded in the E. histolytica genome based on sequence analysis. Using multiple tools, we have analyzed the primary sequences of the eight E. histolytica formins and discovered three subsets: (i) E. histolytica formin-1 to -3 (Ehformin-1 to -3), (ii) Ehformin-4, and (iii) Ehformin-5 to -8. Two of these subsets (Ehformin-1 to -3 and Ehformin-4) showed significant sequence differences from their closest homologs, while Ehformin-5 to -8 were unique among all known formins. Since Ehformin-1 to -3 showed important sequence differences from Diaphanous-related formins (DRFs), we have studied the functions of Ehformin-1 and -2 in E. histolytica transformants. Like other DRFs, Ehformin-1 and -2 associated with F-actin in response to serum factors, in pseudopodia, in pinocytic and phagocytic vesicles, and at cell division sites. Ehformin-1 and -2 also localized with the microtubular assembly in the nucleus, indicating their involvement in genome segregation. While increased expression of Ehformin-1 and -2 did not affect phagocytosis or motility, it clearly showed an increase in the number of binucleated cells, the number of nuclei in multinucleated cells, and the average DNA content of each nucleus, suggesting that these proteins regulate both mitosis and cytokinesis in E. histolytica.

Entamoeba histolytica encodes unique formins, a subset of which regulates DNA content and cell division.

Accumulation of multiple copies of the genome in a single nucleus and several nuclei in a single cell has previously been noted in Entamoeba histolytica, contributing to the genetic heterogeneity of this unicellular eukaryote. In this study, we demonstrate that this genetic heterogeneity is an inherent feature of the cell cycle of this organism. Chromosome segregation occurs on a variety of novel microtubular assemblies including multi-polar spindles. Cytokinesis in E. histolytica is completed by the mechanical severing of a thin cytoplasmic bridge, either independently or with the help of neighboring cells. Importantly, cytokinesis is uncoupled from the nuclear division cycle, both temporally and spatially, leading to the formation of unequal daughter cells. Sorting of euploid and polyploid cells showed that each of these sub-populations acquired heterogeneous DNA content upon further growth. Our study conclusively demonstrates that genetic heterogeneity originates from the unique mode of cell division events in this protist.

Inter-cellular variation in DNA content of Entamoeba histolytica originates from temporal and spatial uncoupling of cytokinesis from the nuclear cycle.

The nondegradable Mps1(Δ12/13) protein drives centriole overproduction, suggesting that Mps1 phosphorylates a subset of centrosomal proteins to drive the assembly of new centrioles. Here we identify three Mps1 phosphorylation sites within the centriolar protein Centrin 2 (Cetn2). Although centrioles can be assembled in the absence of Cetn2, centriole assembly is attenuated in the absence of Cetn2. While wild-type Cetn2 can compensate for this attenuation, a nonphosphorylatable version cannot. In addition, overexpressing Cetn2 causes Mps1-dependent centriole overproduction that requires each of the three Mps1 phosphorylation sites within Cetn2 and is greatly exacerbated by mimicking phosphorylation at any of these sites. Wild-type Cetn2 generates excess foci that are competent as mitotic spindle poles in HsSas-6-depleted cells, suggesting that Cetn2 can organize a subset of centriolar proteins independently of cartwheels. However, centriole overproduction caused by a phosphomimetic Cetn2 mutant requires HsSas-6, suggesting that Cetn2 phosphorylation stimulates the canonical centriole assembly pathway. Moreover, in the absence of Cetn2, Mps1(Δ12/13) cannot drive the production of mature centrioles capable of recruiting γ-Tubulin, and a nonphosphorylatable Cetn2 mutant cannot compensate for this defect and exacerbates Cetn2 depletion. Together, our data suggest that Mps1-dependent phosphorylation of Cetn2 stimulates the canonical centriole assembly pathway.

Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly.

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.

Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase.

Centrioles are duplicated during S-phase to generate the two centrosomes that serve as mitotic spindle poles during mitosis. The centrosomal pool of the Mps1 kinase is important for centriole assembly, but how Mps1 is delivered to centrosomes is unknown. Here we have identified a centrosome localization domain within Mps1 and identified the mitochondrial porin VDAC3 as a protein that binds to this region of Mps1. Moreover, we show that VDAC3 is present at the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes.

VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes.

Centrosomes serve to organize new centrioles in cycling cells, whereas in quiescent cells they assemble primary cilia. We have recently shown that the mitochondrial porin VDAC3 is also a centrosomal protein that is predominantly associated with the mother centriole and modulates centriole assembly by recruiting Mps1 to centrosomes. Here, we show that depletion of VDAC3 causes inappropriate ciliogenesis in cycling cells, while expression of GFP-VDAC3 suppresses ciliogenesis in quiescent cells. Mps1 also negatively regulates ciliogenesis, and the inappropriate ciliogenesis caused by VDAC3 depletion can be bypassed by targeting Mps1 to centrosomes independently of VDAC3. Thus, our data show that a VDAC3-Mps1 module at the centrosome promotes ciliary disassembly during cell cycle entry and suppresses cilia assembly in proliferating cells. Our data also suggests that VDAC3 might be a link between mitochondrial dysfunction and ciliopathies in mammalian cells.

VDAC3 and Mps1 negatively regulate ciliogenesis.

Glioblastoma (GBM) is the most common and lethal primary brain tumor and remains incurable. This is in part due to the cellular heterogeneity within these tumors, which includes a subpopulation of treatment-resistant cells called cancer stem-like cells (CSC). We previously identified that the anaphase-promoting complex/cylosome (APC/C), a key cell-cycle regulator and tumor suppressor, had attenuated ligase activity in CSCs. Here, we assessed the mechanism of reduced activity, as well as the efficacy of pharmacologically targeting the APC/C in CSCs. We identified hyperphosphorylation of CDH1, but not pseudosubstrate inhibition by early mitotic inhibitor 1 (EMI1), as a major mechanism driving attenuated APC/C activity in the G-phase of the cell cycle in CSCs. Small-molecule inhibition of the APC/C reduced viability of both CSCs and nonstem tumor cells (NSTCs), with the combination of proTAME and apcin having the biggest impact. Combinatorial drug treatment also led to the greatest mitotic arrest and chromosomal abnormalities. IMPLICATIONS: Our findings demonstrate how the activity of the APC/C tumor suppressor is reduced in CSCs and also validates small-molecule inhibition of the APC/C as a promising therapeutic target for the treatment of GBM.

Hyperphosphorylation of CDH1 in Glioblastoma Cancer Stem Cells Attenuates APC/C Activity and Pharmacologic Inhibition of APC/C Compromises Viability.

KIF11 is a homotetrameric kinesin that peaks in protein expression during mitosis. It is a known mitotic regulator, and it is well-described that KIF11 is necessary for the formation and maintenance of the bipolar spindle. However, there has been a growing appreciation for non-mitotic roles for KIF11. KIF11 has been shown to function in such processes as axon growth and microtubule polymerization. We previously demonstrated that there is an interphase pool of KIF11 present in glioblastoma cancer stem cells that drives tumor cell invasion. Here, we identified a previously unknown association between KIF11 and primary cilia. We confirmed that KIF11 localized to the basal bodies of primary cilia in multiple cell types, including neoplastic and non-neoplastic cells. Further, we determined that KIF11 has a role in regulating cilia dynamics. Upon the reduction of KIF11 expression, the number of ciliated cells in asynchronously growing populations was significantly increased. We rescued this effect by the addition of exogenous KIF11. Lastly, we found that depleting KIF11 resulted in an increase in cilium length and an attenuation in the kinetics of cilia disassembly. These findings establish a previously unknown link between KIF11 and the dynamics of primary cilia and further support non-mitotic functions for this kinesin.

An interphase pool of KIF11 localizes at the basal bodies of primary cilia and a reduction in KIF11 expression alters cilia dynamics.

Primary cilia are non-motile, microtubule-based, antennae-like organelle that protrude out from the cell surface and perform sensory function or transduce physiological signals in majority of the vertebrate cells. Cilia are assembled on basal bodies that are transformed centrioles. The assembly-disassembly of primary cilia may pose an additional measure on regulating cell cycle in vertebrate cells. While primary cilia are commonly found in differentiated or quiescent cells that are not cycling, disassembly of primary cilia may promote re-entry of these cells into the mitotic cycle, and support proliferation. Many cancer tissues or cancer-derived cells exhibit loss of primary cilia. However, primary cilia may also promote tumorigenesis in some contexts through growth-promoting signalling. This review will shed light on recent advancements of temporal coordination of ciliary disassembly and cell cycle progression, with a focus on how cilia loss may support tumorigenesis in various epithelial cancers.

Freeing the brake: Proliferation needs primary cilium to disassemble.

Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87MG cells significantly increased ciliation. Those PCs were significantly longer than the control cells. Such increased ciliation possibly inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC disassembly and cilia length.

Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

ArticleTotal : 2	Year
Publication title Cited by 14	2014
Publication title Cited by 1	2025

Article	Year
Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica. Cellular microbiology\| PubMed ID: 16925786	2007
EhRho1, a RhoA-like GTPase of Entamoeba histolytica, is modified by clostridial glucosylating cytotoxins. Applied and environmental microbiology\| PubMed ID: 17056697	2006
Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica. Bioscience reports\| PubMed ID: 17592766	2007
Entamoeba histolytica encodes unique formins, a subset of which regulates DNA content and cell division. Infection and immunity\| PubMed ID: 18347041	2008
Inter-cellular variation in DNA content of Entamoeba histolytica originates from temporal and spatial uncoupling of cytokinesis from the nuclear cycle. PLoS neglected tropical diseases\| PubMed ID: 19352422	2009
Mps1 phosphorylation sites regulate the function of centrin 2 in centriole assembly. Molecular biology of the cell\| PubMed ID: 20980622	2010
Ubiquitin ligase Ufd2 is required for efficient degradation of Mps1 kinase. The Journal of biological chemistry\| PubMed ID: 22045814	2011
VDAC3 regulates centriole assembly by targeting Mps1 to centrosomes. Cell cycle (Georgetown, Tex.)\| PubMed ID: 22935710	2012
VDAC3 and Mps1 negatively regulate ciliogenesis. Cell cycle (Georgetown, Tex.)\| PubMed ID: 23388454	2013
Hyperphosphorylation of CDH1 in Glioblastoma Cancer Stem Cells Attenuates APC/C Activity and Pharmacologic Inhibition of APC/C Compromises Viability. Molecular cancer research : MCR\| PubMed ID: 31036696	2019
An interphase pool of KIF11 localizes at the basal bodies of primary cilia and a reduction in KIF11 expression alters cilia dynamics. Scientific reports\| PubMed ID: 32811879	2020
Freeing the brake: Proliferation needs primary cilium to disassemble. Journal of biosciences\| PubMed ID: 33051411	2020
Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells. Experimental cell research\| PubMed ID: 37276998	2023
The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs. Journal of cell science\| PubMed ID: 38841902	2024

Shubhra Majumder

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