Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells

We report the interactions amongst 20 proteins that specify their assembly to the centromere–kinetochore complex in human cells. Centromere protein (CENP)-A is at the top of a hierarchy that directs three major pathways, which are specified by CENP-C, -I, and Aurora B. Each pathway consists of branches that intersect to form nodes that may coordinate the assembly process. Complementary EM studies found that the formation of kinetochore trilaminar plates depends on the CENP-I/NUF2 branch, whereas CENP-C and Aurora B affect the size, shape, and structural integrity of the plates. We found that hMis12 is not constitutively localized at kinetochores, and that it is not essential for recruiting CENP-I. Our studies also revealed that kinetochores in HeLa cells contain an excess of CENP-A, of which ∼10% is sufficient to promote the assembly of normal levels of kinetochore proteins. We elaborate on a previous model that suggested kinetochores are assembled from repetitive modules (Zinkowski, R.P., J. Meyne, and B.R. Brinkley. 1991. J. Cell Biol. 113:1091–110).

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The wages of CIN

The Journal of Cell Biology ◽

10.1083/jcb.200801030 ◽

2008 ◽

Vol 180 (4) ◽

pp. 661-663 ◽

Cited By ~ 12

Author(s):

Karen W. Yuen ◽

Arshad Desai

Keyword(s):

Cancer Cells ◽

Solid Tumors ◽

Chromosomal Instability ◽

Chromosome Instability ◽

Spindle Checkpoint ◽

Human Cells ◽

Normal Cells ◽

Chromosome Missegregation ◽

Cell Biol ◽

The Relationship

Aneuploidy and chromosome instability (CIN) are hallmarks of the majority of solid tumors, but the relationship between them is not well understood. In this issue, Thompson and Compton (Thompson, S.L., and D.A. Compton. 2008. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell. Biol. 180:665–672) investigate the mechanism of CIN in cancer cells and find that CIN arises primarily from defective kinetochore–spindle attachments that evade detection by the spindle checkpoint and persist into anaphase. They also explore the consequences of artificially elevating chromosome missegregation in otherwise karyotypically normal cells. Their finding that induced aneuploidy is rapidly selected against suggests that the persistence of aneuploid cells in tumors requires not only chromosome missegregation but also additional, as yet poorly defined events.

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Injection of Propidium Iodide into HeLa Cells Using a Silicon Nanoinjection Lance Array

Journal of Nanotechnology in Engineering and Medicine ◽

10.1115/1.4028603 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 7

Author(s):

Zachary K. Lindstrom ◽

Steven J. Brewer ◽

Melanie A. Ferguson ◽

Sandra H. Burnett ◽

Brian D. Jensen

Keyword(s):

Gene Therapy ◽

Nucleic Acids ◽

Propidium Iodide ◽

Hela Cells ◽

Human Cells ◽

Advantages And Disadvantages ◽

Culture Cells ◽

High Viability ◽

Promising Area ◽

Hela Cell Culture

Delivering foreign molecules into human cells is a wide and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via nonviral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. We present an electromechanical method for delivering foreign molecules into human cells. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. This paper describes an array of solid silicon microlances that was tested to determine efficiency and viability when nanoinjecting tens of thousands of HeLa cells simultaneously. Propidium iodide (PI), a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers PI into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78–91% viability. With these results, we submit the nanoinjection method using a silicon lance array as another promising particle delivery method for mammalian culture cells.

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Replication and transcription sites are colocalized in human cells

Journal of Cell Science ◽

10.1242/jcs.107.2.425 ◽

1994 ◽

Vol 107 (2) ◽

pp. 425-434 ◽

Cited By ~ 7

Author(s):

A.B. Hassan ◽

R.J. Errington ◽

N.S. White ◽

D.A. Jackson ◽

P.R. Cook

Keyword(s):

Cell Cycle ◽

Confocal Microscopy ◽

Dna Synthesis ◽

Fluorescent Probes ◽

Hela Cells ◽

Nuclear Architecture ◽

Human Cells ◽

Dynamic Nature ◽

Nucleotide Triphosphates ◽

Transcription Sites

HeLa cells synchronized at different stages of the cell cycle were permeabilized and incubated with analogues of nucleotide triphosphates; then sites of incorporation were immunolabeled with the appropriate fluorescent probes. Confocal microscopy showed that sites of replication and transcription were not diffusely spread throughout nuclei, reflecting the distribution of euchromatin; rather, they were concentrated in ‘foci’ where many polymerases act together. Transcription foci aggregated as cells progressed towards the G1/S boundary; later they dispersed and became more diffuse. Replication was initiated only at transcription sites; later, when heterochromatin was replicated in enlarged foci, these remained sites of transcription. This illustrates the dynamic nature of nuclear architecture and suggests that transcription may be required for the initiation of DNA synthesis.

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A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells

Development ◽

10.1242/dev.61382 ◽

2010 ◽

Vol 137 (23) ◽

pp. e2308-e2308

Author(s):

Y. Abe ◽

E. Okumura ◽

T. Hosoya ◽

T. Hirota ◽

...

Keyword(s):

Aurora Kinase ◽

Human Cells ◽

Aurora B ◽

Aurora A

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A single starfish Aurora kinase performs the combined functions of Aurora-A and Aurora-B in human cells

Journal of Cell Science ◽

10.1242/jcs.076315 ◽

2010 ◽

Vol 123 (22) ◽

pp. 3978-3988 ◽

Cited By ~ 15

Author(s):

Y. Abe ◽

E. Okumura ◽

T. Hosoya ◽

T. Hirota ◽

T. Kishimoto

Keyword(s):

Aurora Kinase ◽

Human Cells ◽

Aurora B ◽

Aurora A

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Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

Journal of Virology ◽

10.1128/jvi.80.2.964-974.2006 ◽

2006 ◽

Vol 80 (2) ◽

pp. 964-974 ◽

Cited By ~ 35

Author(s):

Ramon Gonzalez ◽

Wenying Huang ◽

Renee Finnen ◽

Courtney Bragg ◽

S. J. Flint

Keyword(s):

Dna Synthesis ◽

Hela Cells ◽

Human Fibroblasts ◽

Late Phase ◽

Mrna Export ◽

Human Cells ◽

Insertion Mutation ◽

Viral Dna ◽

Normal Human ◽

Normal Human Cells

ABSTRACT The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).

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A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.2072-2081.1984 ◽

1984 ◽

Vol 4 (10) ◽

pp. 2072-2081

Author(s):

J D Tratschin ◽

M H West ◽

T Sandbank ◽

B J Carter

Keyword(s):

Hela Cells ◽

High Efficiency ◽

Constitutive Expression ◽

Chloramphenicol Acetyltransferase ◽

Human Cells ◽

Foreign Gene ◽

Similar Amount ◽

Adeno Associated Virus ◽

293 Cells ◽

Bacterial Plasmid

We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

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Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis

Journal of Cell Science ◽

10.1242/jcs.109.2.277 ◽

1996 ◽

Vol 109 (2) ◽

pp. 277-288 ◽

Cited By ~ 7

Author(s):

H.L. Hsu ◽

N.H. Yeh

Keyword(s):

Cell Cycle ◽

Hela Cells ◽

Structural Integrity ◽

Chromatin Condensation ◽

Tail Domain ◽

Truncated Form ◽

Cdc2 Kinase ◽

Mitotic Apparatus ◽

Interphase Cells ◽

Late Stages

We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phase-arrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.

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Nanoarchitectonics meets cell surface engineering: shape recognition of human cells by halloysite-doped silica cell imprints

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.10.176 ◽

2019 ◽

Vol 10 ◽

pp. 1818-1825 ◽

Cited By ~ 14

Author(s):

Elvira Rozhina ◽

Ilnur Ishmukhametov ◽

Svetlana Batasheva ◽

Farida Akhatova ◽

Rawil Fakhrullin

Keyword(s):

Cell Surface ◽

Hela Cells ◽

Surface Engineering ◽

Human Cells ◽

Live Cells ◽

Liquid Media ◽

Cell Surface Engineering ◽

Produce Cell ◽

Silica Cell ◽

Clay Nanotubes

Cell surface engineering, as a practical manifestation of nanoarchitectonics, is a powerful tool to modify and enhance properties of live cells. In turn, cells may serve as sacrificial templates to fabricate cell-mimicking materials. Herein we report a facile method to produce cell-recognising silica imprints capable of the selective detection of human cells. We used HeLa cells to template silica inorganic shells doped with halloysite clay nanotubes. The shells were destroyed by sonication resulting in the formation of polydisperse hybrid imprints that were used to recognise HeLa cells in liquid media supplemented with yeast. We believe that methodology reported here will find applications in biomedical and clinical research.

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