scholarly journals Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages

2003 ◽  
Vol 160 (5) ◽  
pp. 685-697 ◽  
Author(s):  
Joachim Walter ◽  
Lothar Schermelleh ◽  
Marion Cremer ◽  
Satoshi Tashiro ◽  
Thomas Cremer
Keyword(s):  
Cell Cycle ◽  
Nuclear Architecture ◽  
Metaphase Plate ◽  
Early Prophase ◽  
Chromosome Arrangements ◽  
Variable Extent ◽  
Chromosome Segments ◽  
Chromatin Patterns

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several μm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of ∼1 μm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.

Extracellular Matrix Protein Matrilin-4 Regulates HSC Stress Response

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 601-601
Author(s):  
Hannah Uckelmann ◽  
Sandra Blaszkiewicz ◽  
Marieke Essers
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Bone Marrow Cells ◽  
Blood System ◽  
Extracellular Matrix Protein

Abstract The life-long maintenance of the blood system is accomplished by a pool of self-renewing multipotent hematopoietic stem cells (HSCs). Adult HSCs are found in a dormant state for most of their lifetime, entering cell cycle only to maintain homeostatic blood supply. Under stress conditions such as infection or chemotherapy, the loss of mature blood cells leads to an activation of dormant HSCs to replenish the blood system. Gene expression analysis performed by our group now revealed that Matrilin-4 is highly expressed in long-term HSCs (LT-HSCs) compared to short-term HSCs or committed progenitors, suggesting a potential role of Matrilin-4 in HSC function. Matrilin-4 is a member of the von Willebrand factor A-containing family of extracellular adapter proteins, which form filamentous structures outside of cells. Using mice lacking the entire family of Matrilins (1-4) we have investigated the role of Matrilins in HSC function. Constitutive Matrilin 1-4 KO mice exhibit normal hematopoiesis with a mild reduction in bone marrow cellularity and LSK numbers. However, when Matrilin KO bone marrow cells are pushed to proliferate in competitive transplantation assays with wildtype (WT) cells, they show a striking growth advantage. In a competitive transplant setting, where bone marrow cells of Matrilin KO versus WT mice are transplanted in a 1:1 ratio, the KO cells outcompete WT cells within four weeks, reaching a 90% chimerism at 16 weeks. This competitive advantage of Matrilin KO cells is evident in the long-term stem cell level as well as progenitors and is consistent in secondary transplants. To explore this remarkable phenotype, we performed single cell transplantation experiments of LT-HSCs and observed a more rapid reconstitution of peripheral blood cell levels of KO HSCs compared to WT controls. Confirming this growth advantage, Matrilin KO LSK cells show higher colony forming and serial replating potential in vitro, which can be rescued by the addition of recombinant or overexpressed Matrilin-4. While Matrilin-4 is highly expressed in homeostatic HSCs, in vivo treatment with IFNα or other inflammatory agents, such as LPS or G-CSF result in a dramatic downregulation (25-fold) of Matrilin-4 on the transcript as well as the protein level. Moreover, Matrilin KO HSCs are more sensitive to inflammatory stress, as they show a 2-fold stronger cell cycle activation in response to IFNα in vivo. Critically, Matrilin-4 KO HSCs return to the G0 state of the cell cycle normally after stress-induced activation and transplantation, thereby preventing their exhaustion. In summary, we show that the extracellular matrix protein Matrilin-4 is a novel component of the HSC niche, regulating HSC stress response. Surprisingly, HSCs lacking this extracellular matrix protein show a higher HSC potential due to an accelerated response to stress. Our data suggest that high expression of Matrilin-4 in LT-HSCs confers a resistance to stress stimuli. In situations of acute stress such as infection or transplantation however, this protection is rapidly lost to allow HSCs to efficiently replenish the blood system. Disclosures No relevant conflicts of interest to declare.

scholarly journals Silica Particles Trigger Pulmonary Fibrosis Act Through Cell-To-Cell Delivery of Exosomal Mir-107

2021 ◽  
Author(s):  
Di Wang ◽  
Changfu Hao ◽  
Wei Guo ◽  
Yangqing Pei ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Pulmonary Fibrosis ◽  
Cell Cycle Progression ◽  
Silica Particles ◽  
Lung Fibroblasts ◽  
Targeted Inhibition ◽  
Exosomal Mirna ◽  
Potential Interactions

Abstract Background: Long-term exposure to inhalable silica particles may lead to a serious systemic pulmonary disease called silicosis. However, the role and mechanisms of exosomes in silicosis are not well understood. We previously reported that serum exosomal micro (mi) RNA profile was altered in pneumoconiosis patients and silica-exposed macrophages.This study was aimed to explore and verify the role of the exosomal miRNA in lung fibrosis when exposed to silica particles. Results: The RT-qPCR result revealed that the levels of the miR-107, miR-122-5p, miR-125a-5p, miR-126-5p, and miR-335-5p were elevated in serous exosomes of silicosis patients. A bioinformatics analysis predicted 5 potential interactions involving these miRNAs, with miR-107–cyclin-dependent kinase (CDK) 6 having the highest score. In a mouse model of silica particle-induced silicosis, miR-107 level in serum exosomes and lung tissue was increased during the development of fibrosis, while inhibition of miR-107 reduced pulmonary fibrosis. The number of exosomes secreted by macrophages exposed to silica particles was also increased and showed altered cargo composition, and showed a capacity to promote lung fibroblast transdifferentiation through a possible mechanism involving the delivery of miR-107 by macrophages to lung fibroblasts via exosomes, resulting in targeted inhibition of CDK6, reduced retinoblastoma protein phosphorylation, and inhibition of E2F1 and cell cycle progression. Conclusion: In summary, exosomal miR-107 derived from macrophages exposed to silica particles were transferred to pulmonary fibroblasts to trigger their transdifferentiation by targeting CDK6 and arresting cell cycle. These findings provide insight into the pathogenesis of silicosis and potential targets for intervention.

Cdkn3 Knockout Mice Develop Hematopoietic Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1537-1537 ◽  
Author(s):  
Zejin Sun ◽  
Donna Cerabona ◽  
Ying He ◽  
Grzegorz Nalepa
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Conditional Knockout ◽  
Cycle Progression ◽  
Mitotic Kinase ◽  
Risk Of Death

Abstract Cyclin dependent kinase inhibitor 3 (CDKN3) is a dual-specificity cell cycle regulatory phosphatase. In interphase, CDKN3 prevents premature G1/S transition by dephosphorylating interphase cyclin-dependent kinases (CDKs) to prevent premature inactivation of the RB pathway. During cell division, CDKN3 dephosphorylates the key mitotic kinase CDK1 at threonine-161 to extinguish CDK1 activity at the exit from mitosis. CDKN3 knockdown in cultured cells impairs the spindle assembly checkpoint (SAC), accelerates cell cycle progression and causes chromosomal instability, suggesting that it may function as a tumor suppressor. However, since CDKN3 has been reported as overexpressed in some malignancies and mutated or silenced in others, it is unclear whether it functions as an oncogene or a tumor suppressor. To understand the in vivo role of CDKN3 in carcinogenesis, we generated the first Cdkn3 conditional knockout mouse model. We found that Cdkn3-/- mice were viable, non-dysmorphic and born at expected Mendelian ratios, indicating that this gene is dispensable for normal embryonic development. In agreement with the postulated role of this phosphatase in cell cycle progression and regulation of CDKs, we found that Cdkn3-/- cells had increased CDK1, CDK2 and CDK4 activity; increased inhibitory phosphorylation of Rb; increased DNA replication and proliferation; and impaired SAC. Increased CDK activity and accelerated cell cycle progression caused genomic instability reflected by increased frequency of in vivo micronucleation during hematopoiesis as well as higher frequency of aneuploidy and multinucleation and accumulation of supernumerary centrosomes in Cdkn3-/- cells cultured ex vivo. Cdkn3-/- mice had increased myeloid colony-forming units in progenitor assays. Long-term observation of Cdkn3-/- mice revealed an increased risk of death from a variety of hematopoietic (leukemia and lymphoma) and non-hematopoietic (lung, prostate and ovarian) malignancies. Our findings establish Cdkn3 as an in vivo tumor suppressor in bone marrow and a variety of other tissues. In the long term, Cdkn3-/- mice will serve as a tool to dissect the function of this phosphatase in cell cycle control in more detail, and may prove useful in preclinical studies of chemotherapy of CDK-hyperactive, genomically unstable leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Indispensable role of Galectin-3 in promoting quiescence of hematopoietic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weizhen Jia ◽  
Lingyu Kong ◽  
Hiroyasu Kidoya ◽  
Hisamichi Naito ◽  
Fumitaka Muramatsu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transcriptional Control ◽  
Negative Regulator ◽  
Hematopoietic Stem ◽  
Cell Cycle Entry ◽  
Galectin 3

AbstractHematopoietic stem cells (HSCs) in adult bone marrow (BM) are usually maintained in a state of quiescence. The cellular mechanism coordinating the balance between HSC quiescence and differentiation is not fully understood. Here, we report that galactose-binding lectin-3 (galectin-3; Gal-3) is upregulated by Tie2 or Mpl activation to maintain quiescence. Conditional overexpression of Gal-3 in mouse HSCs under the transcriptional control of Tie2 or Vav1 promoters (Gal-3 Tg) causes cell cycle retardation via induction of p21. Conversely, the cell cycle of long-term repopulating HSCs (LT-HSCs) in Gal-3-deficient (Gal-3-/-) mice is accelerated, resulting in their exhaustion. Mechanistically, Gal-3 regulates p21 transcription by forming a complex with Sp1, thus blocking cell cycle entry. These results demonstrate that Gal-3 is a negative regulator of cell-cycling in HSCs and plays a crucial role in adult hematopoiesis to prevent HSC exhaustion.

scholarly journals NRF2 Activation Impairs Quiescence and Bone Marrow Reconstitution Capacity of Hematopoietic Stem Cells

2017 ◽  
Vol 37 (19) ◽  
Author(s):  
Shohei Murakami ◽  
Takuma Suzuki ◽  
Hideo Harigae ◽  
Paul-Henri Romeo ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Cycle Entry ◽  
Transient Activation ◽  
Nrf2 Activation

ABSTRACT Tissue stem cells are maintained in quiescence under physiological conditions but proliferate and differentiate to replenish mature cells under stressed conditions. The KEAP1-NRF2 system plays an essential role in stress response and cytoprotection against redox disturbance. To clarify the role of the KEAP1-NRF2 system in tissue stem cells, we focused on hematopoiesis in this study and used Keap1-deficient mice to examine the effects of persistent activation of NRF2 on long-term hematopoietic stem cells (LT-HSCs). We found that persistent activation of NRF2 due to Keap1 deficiency did not change the number of LT-HSCs but reduced their quiescence in steady-state hematopoiesis. During hematopoietic regeneration after bone marrow (BM) transplantation, persistent activation of NRF2 reduced the BM reconstitution capacity of LT-HSCs, suggesting that NRF2 reduces the quiescence of LT-HSCs and promotes their differentiation, leading to eventual exhaustion. Transient activation of NRF2 by an electrophilic reagent also promotes the entry of LT-HSCs into the cell cycle. Taken together, our findings show that NRF2 drives the cell cycle entry and differentiation of LT-HSCs at the expense of their quiescence and maintenance, an effect that appears to be beneficial for prompt recovery from blood loss. We propose that the appropriate control of NRF2 activity by KEAP1 is essential for maintaining HSCs and guarantees their stress-induced regenerative response.

scholarly journals Conservation of the Centromere/Kinetochore Protein ZW10

1997 ◽  
Vol 138 (6) ◽  
pp. 1289-1301 ◽  
Author(s):  
Daniel A. Starr ◽  
Byron C. Williams ◽  
Zexiao Li ◽  
Bijan Etemad-Moghadam ◽  
R. Kelly Dawe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Metaphase Plate ◽  
C Elegans ◽  
Anaphase Onset ◽  
A Cell ◽  
Cycle Dependent ◽  
Related Proteins ◽  
Drosophila Cells

Mutations in the essential Drosophila melanogaster gene zw10 disrupt chromosome segregation, producing chromosomes that lag at the metaphase plate during anaphase of mitosis and both meiotic divisions. Recent evidence suggests that the product of this gene, DmZW10, acts at the kinetochore as part of a tension-sensing checkpoint at anaphase onset. DmZW10 displays an intriguing cell cycle–dependent intracellular distribution, apparently moving from the centromere/kinetochore at prometaphase to kinetochore microtubules at metaphase, and back to the centromere/kinetochore at anaphase (Williams, B.C., M. Gatti, and M.L. Goldberg. 1996. J. Cell Biol. 134:1127-1140). We have identified ZW10-related proteins from widely diverse species with divergent centromere structures, including several Drosophilids, Caenorhabditis elegans, Arabidopsis thaliana, Mus musculus, and humans. Antibodies against the human ZW10 protein display a cell cycle–dependent staining pattern in HeLa cells strikingly similar to that previously observed for DmZW10 in dividing Drosophila cells. Injections of C. elegans ZW10 antisense RNA phenocopies important aspects of the mutant phenotype in Drosophila: these include a strong decrease in brood size, suggesting defects in meiosis or germline mitosis, a high percentage of lethality among the embryos that are produced, and the appearance of chromatin bridges at anaphase. These results indicate that at least some aspects of the functional role of the ZW10 protein in ensuring proper chromosome segregation are conserved across large evolutionary distances.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2617-2617
Author(s):  
Fumio Arai ◽  
Kentaro Hosokawa ◽  
Yumiko Nojima ◽  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2617 Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs. On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004). In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT. These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures: No relevant conflicts of interest to declare.

In vivo trafficking, cell cycle activity, and engraftment potential of phenotypically defined primitive hematopoietic cells after transplantation into irradiated or nonirradiated recipients

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3545-3552 ◽  
Author(s):  
P. Artur Plett ◽  
Stacy M. Frankovitz ◽  
Christie M. Orschell-Traycoff
Keyword(s):  
Cell Cycle ◽  
Active Role ◽  
Brdu Incorporation ◽  
Recent Interest ◽  
Cell Cycle Status ◽  
In Vivo Recovery ◽  
Serial Transplantation

Recent interest in bone marrow (BM) transplantation in nonconditioned or minimally conditioned recipients warrants investigation of homing patterns of transplanted hematopoietic progenitor cells (HPCs) in irradiated and nonirradiated recipients. To this end, phenotypically defined populations of BM cells were tracked in lethally irradiated or nonirradiated mice at 1, 3, 6, and 24 hours after transplantation. Recovery of transplanted cells at all time points was higher in BM of nonirradiated mice, similar to earlier suggestions. The percentage of lineage-negative Sca-1+cells and Sca-1+ cells expressing CD43, CD49e, and CD49d steadily increased in BM of nonirradiated mice up to 24 hours, while fluctuating in irradiated mice. Cell cycle status and BrdU incorporation revealed that less than 20% of Sca-1+ cells and fewer Sca-1+lin− cells had cycled by 24 hours after transplantation. To more directly examine trafficking of primitive HPCs, purified grafts of CD62L− or CD49e+ subfractions of Sca-1+lin−cells, previously shown to be enriched for long-term repopulating cells, also were tracked in vivo. Recovery of purified cells was similarly increased in BM of nonirradiated mice. When 50 to 100 of these BM-homed cells were examined in serial transplantation studies, BM-homed cells from initially nonirradiated mice were enriched 5- to 30-fold for cells capable of long-term hematopoiesis in secondary recipients. Collectively, these data suggest that homing or survival of transplanted cells in irradiated recipients is less efficient than that in nonirradiated recipients, implicating an active role of radiation-sensitive microenvironmental cues in the homing process. These results may have important clinical implications in the design of BM transplantation protocols.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

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