Iron-(Fe3+)-Dependent Reactivation of Telomerase Drives Colorectal Cancers.

Over-consumption of iron-rich red meat and hereditary or genetic iron overload are associated with an increased risk of colorectal carcinogenesis, yet the mechanistic basis of how metal-mediated signaling leads to oncogenesis remains enigmatic. Using fresh colorectal cancer samples we identify Pirin, an iron sensor, that overcomes a rate-limiting step in oncogenesis, by reactivating the dormant human telomerase reverse transcriptase (hTERT) subunit of the telomerase holoenzyme in an iron-(Fe3+)-dependent manner and thereby drives colorectal cancers. Chemical genetic screens combined with isothermal dose-response fingerprinting and mass spectrometry identified a small molecule SP2509 that specifically inhibits Pirin-mediated hTERT reactivation in colorectal cancers by competing with iron-(Fe3+) binding. Our findings, first to document how metal ions reactivate telomerase, provide a molecular mechanism for the well-known association between red meat and increased incidence of colorectal cancers. Small molecules like SP2509 represent a novel modality to target telomerase that acts as a driver of 90% of human cancers and is yet to be targeted in clinic. Significance: We show how iron-(Fe3+) in collusion with genetic factors reactivates telomerase, providing a molecular mechanism for the association between iron overload and increased incidence of colorectal cancers. Although no enzymatic inhibitors of telomerase have entered the clinic, we identify SP2509, a small molecule that targets telomerase reactivation and function in colorectal cancers.

Histones to the cytosol: exportin 7 is essential for normal terminal erythroid nuclear maturation.

Global nuclear condensation, culminating in enucleation during terminal erythropoiesis, is poorly understood. Proteomic examination of extruded erythroid nuclei from fetal liver revealed a striking depletion of most nuclear proteins, suggesting that nuclear protein export had occurred. Expression of the nuclear export protein, Exportin 7 (Xpo7), is highly erythroid-specific, induced during erythropoiesis, and abundant in very late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe inhibition of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation. Nuclei in Xpo7-knockdown cells were larger and less dense than normal and accumulated most nuclear proteins as measured by mass spectrometry. Strikingly,many DNA binding proteins such as histones H2A and H3 were found to have migrated into the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in Xpo7-knockdown cells. Thus, terminal erythroid maturation involves migration of histones into the cytoplasm via a process likely facilitated by Xpo7.

The rice TAL effector-dependent resistance protein XA10 triggers cell death and calcium depletion in the endoplasmic reticulum.

The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene-mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator-like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca(2+) depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca(2+) depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca(2+) homeostasis.

Proper positioning of the cleavage furrow requires α-actinin to regulate the specification of different populations of microtubules.

Proper positioning of the cleavage furrow is essential for successful cell division. The mitotic spindle, which consists of dynamic astral microtubules and stable equatorial microtubules is responsible for this process. However, little is known about how microtubules are regulated in a time- and region-dependent manner. Here, we show that α-actinin-regulated cortical actin filament integrity is crucial to specify different populations of microtubules during cell division in mammalian cells. Depletion of α-actinin caused aberrant recruitment of centralspindlin, but not aurora B or PRC1, to the tips of astral microtubules, leading to a stable association of astral microtubules with the cortex and induction of ectopic furrowing. Depletion of α-actinin also caused impaired assembly of midzone microtubules, leading to a failure of relocation of aurora B to midzone. Our findings unveil an unexpected yet crucial role for an actin crosslinking protein in the regulation of the localization of the microtubule-associated cytokinetic regulator.

RUNX3 interactome reveals novel centrosomal targeting of RUNX family of transcription factors.

RUNX family proteins are critical regulators of lineage differentiation during development. The high prevalence of RUNX mutation/epigenetic inactivation in human cancer indicates a causative role for dysfunctional RUNX in carcinogenesis. This is supported by well-documented evidence of functional interaction of RUNX with components of major oncogenic or tumor suppressive signaling pathways such as TGFß and Wnt. Here, we explore the binding partners of RUNX3 proteins to further define the scope of RUNX3 function. Using a mass spectrometry-based approach, we found that RUNX3 binds to centrosomal protein rootletin. This led us to uncover the presence of RUNX proteins at the centrosome. Our findings suggest a potential function for RUNX3 during mitosis.

Calcium sensitivity of α-actinin is required for equatorial actin assembly during cytokinesis.

The actin cross-linking protein, α-actinin, plays a crucial role in mediating furrow ingression during cytokinesis. However, the mechanism by which its dynamics are regulated during this process is poorly understood. Here we have investigated the role of calcium sensitivity of α-actinin in the regulation of its dynamics by generating a functional calcium-insensitive mutant (EFM). GFP-tagged EFM (EFM-GFP) localized to the equatorial regions during cell division. However, the maximal equatorial accumulation of EFM-GFP was significantly smaller in comparison to α-actinin-GFP when it was expressed in normal cells and cells depleted of endogenous α-actinin. No apparent defects in cytokinesis were observed in these cells. However, F-actin levels at the equator were significantly reduced in cells expressing EFM-GFP as compared with α-actinin-GFP at furrow initiation but were recovered during furrow ingression. These results suggest that calcium sensitivity of α-actinin is required for its equatorial accumulation that is crucial for the initial equatorial actin assembly but is dispensable for cytokinesis. Equatorial RhoA localization was not affected by EFM-GFP overexpression, suggesting that equatorial actin assembly is predominantly driven by the RhoA-dependent mechanism. Our observations shed new light on the role and regulation of the accumulation of pre-existing actin filaments in equatorial actin assembly during cytokinesis.

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.

HURP regulates chromosome congression by modulating kinesin Kif18A function.

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. The dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin ß inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.

Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

Formation of mammalian erythrocytes: chromatin condensation and enucleation.

In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell-matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast chromatin condensation and enucleation, and conclude with our perspectives on future studies.

Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts.

BACKGROUND: During the final stages of differentiation of mammalian erythroid cells, the chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. DESIGN AND METHODS: Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short hairpin RNA. RESULTS: We showed that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked chromatin condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of chromatin condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. CONCLUSIONS: These results identify histone deacetylase 2 as an important regulator, mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis.

Mitotic Golgi vesiculation involves mechanisms independent of Ser25 phosphorylation of GM130.

During mitosis, the Golgi undergoes two sequential fragmentation steps to break from ribbon to individual stacks, then from stacks to vesicles. While the mechanism that regulates the first step has been studied, it remains obscure how the second vesiculation step is regulated. It has been suggested that Cdk1-dependent phosphorylation of the cis-Golgi matrix protein GM130 regulates the second step. Here we have tested if phorphorylation of GM130 by Cdk1 is required for Golgi vesiculation and mitotic progression. Inhibition of Cdk1 activity caused a failure of Golgi vesiculation and defects in chromosome congression/segregation. Expression of non-phosphorylatable mutant of GM130 (GM130S25A) in cells depleted of endogenous GM130 caused no apparent defects in Golgi vesiculation and mitotic progression. Similarly, no apparent defects in Golgi vesiculation and mitotic progression were observed when GM130S25A was expressed in GM130-deficient CHO cells. Our observations suggest that while Cdk1 based phosphorylation is essential for mitotic Golgi vesiculation, mammalian cells could possess redundant, S25 phosphorylation of GM130 independent pathways that ensure Golgi vesiculation and mitotic progression.

Domain analysis of alpha-actinin reveals new aspects of its association with F-actin during cytokinesis.

alpha-Actinin is a rod-shaped actin cross-linking protein composed of actin binding domain, spectrin-like repeats of the central rod domain and the EF-hand domain. Cytokinesis in mammalian cells involves remodeling of equatorial actin filaments (F-actin) mediated by alpha-actinin. However, it remains unknown how alpha-actinin interacts with F-actin at the cleavage furrow. To address this question, we have conducted functional analysis of the mutant that either lacks the ability to cross-link F-actin (ABD) or to bind to F-actin (DeltaABD). We found that equatorial localization of alpha-actinin requires both its F-actin binding and cross-linking activities. Unexpectedly, we also found that overexpression of DeltaABD-GFP but not ABD-GFP frequently caused accelerated cytokinesis and ectopic furrowing similar to those observed in cells depleted of alpha-actinin. Immunofluorescence revealed that overexpression of DeltaABD-GFP caused displacement of endogenous alpha-actinin and a decrease in the density of F-actin throughout the entire cortex. Biochemical experiments showed that DeltaABD was able to form heterodimers with endogenous alpha-actinin. These results suggest that the central rod spectrin-like repeats of alpha-actinin is sufficient for its dimerization in vivo. Our findings uncover previously unappreciated functions of the alpha-actinin domains in a cell.

Alpha-actinin is required for tightly regulated remodeling of the actin cortical network during cytokinesis.

Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.

Aurora B kinase activity is required to prevent polar cortical ingression during cytokinesis.

Cytokinesis requires proper regulation of microtubule dynamics. It has been suggested that dynamic astral microtubules prevent cortical ingression. However, it remains unknown how astral microtubules maintain their dynamic state. Here we show that aurora B kinase, a component of the chromosome passenger complex, is required to sustain the dynamic state of astral microtubules during cytokinesis. Treatment of HeLa cells with Hesperadin, an inhibitor of aurora B kinase, caused abnormal cortical protrusion, leading to cortical ingression in the protruding region and cytokinesis failure. Actin filaments, myosin II, and RhoA failed to localize at the equator but instead distributed along the lateral and/or polar cortex in cells treated with Hesperadin. Time-lapse analyses of microtubule dynamics showed that, in cells treated with Hesperadin, abnormally bundled astral microtubules targeted the protruding region. Mitotic kinesin-like protein 1 (MKLP1), a component of the spindle midzone required for bundling of microtubules, was not detected along bundled astral microtubules in cells treated with Hesperadin, suggesting that factors other than MKLP1 may be involved in this process. Our results suggest that aurora B kinase activity is required for proper regulation of microtubule dynamics to ensure that cytokinesis occurs precisely at the cell equator.

Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

Specific distribution of overexpressed aurora B kinase during interphase of normal epithelial cells.

BACKGROUND: It is known that aurora B, a chromosomal passenger protein responsible for the proper progression of mitosis and cytokinesis, is overexpressed throughout the cell cycle in cancer cells. Overexpression of aurora B produced multinuclearity and induced aggressive metastasis, suggesting that overexpressed aurora B has multiple functions in cancer development. However, the detailed dynamics and functions of overexpressed aurora B are poorly understood. RESULTS: We overexpressed GFP fused aurora B kinase in normal rat kidney epithelial cells. Using spinning disk confocal microscopy, we found that overexpressed aurora B-GFP was predominantly localized in the nucleus and along the cortex as a dot-like or short filamentous structure during interphase. Time-lapse imaging revealed that a cytoplasmic fraction of overexpressed aurora B-GFP was incorporated into the nucleus after cell division. Immunofluorescence showed that the nuclear fraction of overexpressed aurora B did not induce ectopic phosphorylation of histone H3 after cell division. The cytoplasmic fraction of overexpressed aurora B-GFP was mainly associated with cortical actin filaments but not stress fibers. Myosin II regulatory light chain, one of the possible targets for aurora B, did not colocalize with cortical aurora B-GFP, suggesting that overexpressed aurora B did not promote phosphorylation of myosin II regulatory light chain in interphase cells. CONCLUSION: We conclude that overexpressed aurora B has a specific localization pattern in interphase cells. Based on our findings, we propose that overexpressed aurora B targets the nuclear and cortical proteins during interphase, which may contribute to cancer development and tumor metastasis.

Regulation of cell cycle by the anaphase spindle midzone.

BACKGROUND: A number of proteins accumulate in the spindle midzone and midbody of dividing animal cells. Besides proteins essential for cytokinesis, there are also components essential for interphase functions, suggesting that the spindle midzone and/or midbody may play a role in regulating the following cell cycle. RESULTS: We microsurgically severed NRK epithelial cells during anaphase or telophase, such that the spindle midzone/midbody was associated with only one of the daughter cells. Time-lapse recording of cells severed during early anaphase indicated that the cell with midzone underwent cytokinesis-like cortical contractions and progressed normally through the interphase, whereas the cell without midzone showed no cortical contraction and an arrest or substantial delay in the progression of interphase. Similar microsurgery during telophase showed a normal progression of interphase for both daughter cells with or without the midbody. Microsurgery of anaphase cells treated with cytochalasin D or nocodazole indicated that interphase progression was independent of cortical ingression but dependent on microtubules. CONCLUSIONS: We conclude that the mitotic spindle is involved in not only the separation of chromosomes but also the regulation of cell cycle. The process may involve activation of components in the spindle midzone that are required for the cell cycle, and/or degradation of components that are required for cytokinesis but may interfere with the cell cycle.

Identification and characterization of novel components of a Ca2+/calmodulin-dependent protein kinase cascade in HeLa cells.

In this report, we cloned a novel calmodulin-kinase (CaM-KIdelta) from HeLa cells and characterized its activation mechanism. CaM-KIdelta exhibits Ca(2+)/CaM-dependent activity that is enhanced (approximately 30-fold) in vitro by phosphorylation of its Thr180 by CaM-K kinase (CaM-KK)alpha, consistent with detection of CaM-KIdelta-activating activity in HeLa cells. We also identified a novel CaM-KKbeta isoform (CaM-KKbeta-3) in HeLa cells whose activity was highly Ca(2+)/CaM-independent. Transiently expressed CaM-KIdelta exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM-KIdelta was completely abolished by Thr180Ala mutation and inhibited by CaM-KK inhibitor, STO-609, indicating a functional CaM-KK/CaM-KIdelta cascade in HeLa cells.