Positively Charged Tags Impede Protein Mobility in Cells as Quantified by 19F NMR

2019 ◽  
Vol 123 (21) ◽  
pp. 4527-4533 ◽  
Author(s):  
Yansheng Ye ◽  
Qiong Wu ◽  
Wenwen Zheng ◽  
Bin Jiang ◽  
Gary J. Pielak ◽  
...  
Keyword(s):  
19F Nmr ◽  
Protein Mobility ◽  
Positively Charged ◽  
In Cells

scholarly journals The Ndc80 complex uses a tripartite attachment point to couple microtubule depolymerization to chromosome movement

2011 ◽  
Vol 22 (8) ◽  
pp. 1217-1226 ◽  
Author(s):  
John G. Tooley ◽  
Stephanie A. Miller ◽  
P. Todd Stukenberg
Keyword(s):  
Point Mutations ◽  
Chromosome Movement ◽  
Microtubule Depolymerization ◽  
Attachment Point ◽  
Binding Interface ◽  
Binding Data ◽  
Ndc80 Complex ◽  
Positively Charged ◽  
In Cells

In kinetochores, the Ndc80 complex couples the energy in a depolymerizing microtubule to perform the work of moving chromosomes. The complex directly binds microtubules using an unstructured, positively charged N-terminal tail located on Hec1/Ndc80. Hec1/Ndc80 also contains a calponin homology domain (CHD) that increases its affinity for microtubules in vitro, yet whether it is required in cells and how the tail and CHD work together are critical unanswered questions. Human kinetochores containing Hec1/Ndc80 with point mutations in the CHD fail to align chromosomes or form productive microtubule attachments. Kinetochore architecture and spindle checkpoint protein recruitment are unaffected in these mutants, and the loss of CHD function cannot be rescued by removing Aurora B sites from the tail. The interaction between the Hec1/Ndc80 CHD and a microtubule is facilitated by positively charged amino acids on two separate regions of the CHD, and both are required for kinetochores to make stable attachments to microtubules. Chromosome congression in cells also requires positive charge on the Hec1 tail to facilitate microtubule contact. In vitro binding data suggest that charge on the tail regulates attachment by directly increasing microtubule affinity as well as driving cooperative binding of the CHD. These data argue that in vertebrates there is a tripartite attachment point facilitating the interaction between Hec1/Ndc80 and microtubules. We discuss how such a complex microtubule-binding interface may facilitate the coupling of depolymerization to chromosome movement.

Fusion of Plant Protoplasts Induced by a Positively Charged Synthetic Phospholipid

1979 ◽  
Vol 34 (5-6) ◽  
pp. 460-462 ◽  
Author(s):  
Toshiyuki Nagata ◽  
Hansjörg Eibl ◽  
Georg Melchers
Keyword(s):  
Cell Fusion ◽  
Chemical Structure ◽  
Positive Charge ◽  
Fusion Activity ◽  
Plant Protoplasts ◽  
Positively Charged ◽  
In Cells

Abstract A newly synthesized phospholipid, 1,2-O-Dipentadecylmethylidene-glycerol-3-phosphoryl-(N-ethyl-amino) -ethanolamine, having positive charge was found to exercise fusion activity on plant proto­ plasts. This phospholipid is not found in cells and has an unusal chemical structure with a dioxolane ring. Some properties of this compound are discussed in relation to phospholipid-induced cell fusion.

Delivery, stabilization, and spatiotemporal activation of cargo molecules in cells with positively charged nanoparticles

2012 ◽  
Vol 48 (93) ◽  
pp. 11461 ◽  
Author(s):  
Shuhei Murayama ◽  
Taihei Nishiyama ◽  
Kaihei Takagi ◽  
Fumi Ishizuka ◽  
Tomofumi Santa ◽  
...  
Keyword(s):  
Charged Nanoparticles ◽  
Positively Charged ◽  
In Cells

Quantification of size effect on protein rotational mobility in cells by 19F NMR spectroscopy

2017 ◽  
Vol 410 (3) ◽  
pp. 869-874 ◽  
Author(s):  
Yansheng Ye ◽  
Qiong Wu ◽  
Wenwen Zheng ◽  
Bin Jiang ◽  
Gary J. Pielak ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Size Effect ◽  
19F Nmr ◽  
Rotational Mobility ◽  
19F Nmr Spectroscopy ◽  
In Cells

scholarly journals The NDC80 complex proteins Nuf2 and Hec1 make distinct contributions to kinetochore–microtubule attachment in mitosis

2011 ◽  
Vol 22 (6) ◽  
pp. 759-768 ◽  
Author(s):  
Lynsie J.R. Sundin ◽  
Geoffrey J. Guimaraes ◽  
Jennifer G. DeLuca
Keyword(s):  
Structural Studies ◽  
Tail Domain ◽  
Wild Type ◽  
Timely Manner ◽  
Microtubule Attachment ◽  
Ndc80 Complex ◽  
Spindle Microtubules ◽  
Mutant Cells ◽  
Positively Charged ◽  
In Cells

Successful mitosis requires that kinetochores stably attach to the plus ends of spindle microtubules. Central to generating these attachments is the NDC80 complex, made of the four proteins Spc24, Spc25, Nuf2, and Hec1/Ndc80. Structural studies have revealed that portions of both Hec1 and Nuf2 N termini fold into calponin homology (CH) domains, which are known to mediate microtubule binding in certain proteins. Hec1 also contains a basic, positively charged stretch of amino acids that precedes its CH domain, referred to as the “tail.” Here, using a gene silence and rescue approach in HeLa cells, we show that the CH domain of Hec1, the CH domain of Nuf2, and the Hec1 tail each contributes to kinetochore–microtubule attachment in distinct ways. The most severe defects in kinetochore–microtubule attachment were observed in cells rescued with a Hec1 CH domain mutant, followed by those rescued with a Hec1 tail domain mutant. Cells rescued with Nuf2 CH domain mutants, however, generated stable kinetochore–microtubule attachments but failed to generate wild-type interkinetochore tension and failed to enter anaphase in a timely manner. These data suggest that the CH and tail domains of Hec1 generate essential contacts between kinetochores and microtubules in cells, whereas the Nuf2 CH domain does not.

Protamine-DNA interaction

1978 ◽  
Vol 36 (2) ◽  
pp. 200-201
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Small Volume ◽  
Double Helix ◽  
Dna Interaction ◽  
Dark Field ◽  
Sperm Head ◽  
Nuclear Proteins ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Dna Double Helix ◽  
Positively Charged

Dark field electron microscopy has been used for the study of the structure of individual macromolecules with a resolution to at least the 5Å level. The use of this technique has been extended to the investigation of structure of interacting molecules, particularly the interaction between DNA and fish protamine, a class of basic nuclear proteins of molecular weight 4,000 daltons.Protamine, which is synthesized during spermatogenesis, binds to chromatin, displaces the somatic histones and wraps up the DNA to fit into the small volume of the sperm head. It has been proposed that protamine, existing as an extended polypeptide, winds around the minor groove of the DNA double helix, with protamine's positively-charged arginines lining up with the negatively-charged phosphates of DNA. However, viewing protamine as an extended protein is inconsistent with the results obtained in our laboratory.

Cytoplasmic Tubules in Cells Infected with Laryngotracheitis Virus

1969 ◽  
Vol 27 ◽  
pp. 376-377
Author(s):  
A. M. Watrach
Keyword(s):  
Tissue Culture ◽  
Small Proportion ◽  
Chicken Embryo ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Morphologic Characteristics ◽  
Infectious Laryngotracheitis ◽  
Laryngotracheitis Virus ◽  
In Cells

During a study of the development of infectious laryngotracheitis (LT) virus in tissue culture cells, unusual tubular formations were found in the cytoplasm of a small proportion of the affected cells. It is the purpose of this report to describe the morphologic characteristics of the tubules and to discuss their possible association with the development of virus.The source and maintenance of the strain of LT virus have been described. Prior to this study, the virus was passed several times in chicken embryo kidney (CEK) tissue culture cells.

Effect of Some Metabolic Inhibitors on Vinblastine-Induced Ribosomal-Granular Material Complexes

1971 ◽  
Vol 29 ◽  
pp. 548-549
Author(s):  
Awtar Krishan ◽  
Dora Hsu
Keyword(s):  
Granular Material ◽  
Rna Synthesis ◽  
Culture Medium ◽  
Actinomycin D ◽  
Metabolic Inhibitors ◽  
Protein And Rna Synthesis ◽  
Apparent Reduction ◽  
Plant Alkaloids ◽  
In Cells

Cells exposed to antitumor plant alkaloids, vinblastine and vincristine sulfate have large proteinacious crystals and complexes of ribosomes, helical polyribosomes and electron-dense granular material (ribosomal complexes) in their cytoplasm, Binding of H3-colchicine by the in vivo crystals shows that they contain microtubular proteins. Association of ribosomal complexes with the crystals suggests that these structures may be interrelated.In the present study cultured human leukemic lymphoblasts (CCRF-CEM), were incubated with protein and RNA-synthesis inhibitors, p. fluorophenylalanine, puromycin, cycloheximide or actinomycin-D before the addition of crystal-inducing doses of vinblastine to the culture medium. None of these compounds could completely prevent the formation of the ribosomal complexes or the crystals. However, in cells pre-incubated with puromycin, cycloheximide, or actinomycin-D, a reduction in the number and size of the ribosomal complexes was seen. Large helical polyribosomes were absent in the ribosomal complexes of cells treated with puromycin, while in cells exposed to cycloheximide, there was an apparent reduction in the number of ribosomes associated with the ribosomal complexes (Fig. 2).

Structure and Interaction of Protamine by Dark Field Electron Microscopy

1976 ◽  
Vol 34 ◽  
pp. 160-161
Author(s):  
D.P. Bazett-Jones ◽  
F.P. Ottensmeyer
Keyword(s):  
Electron Microscopy ◽  
Dark Field ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
3 Dimensional ◽  
Field Electron ◽  
Proposed Model ◽  
Field Electron Microscopy ◽  
Dark Field Electron ◽  
Positively Charged

It has been shown for some time that it is possible to obtain images of small unstained proteins, with a resolution of approximately 5Å using dark field electron microscopy (1,2). Applying this technique, we have observed a uniformity in size and shape of the 2-dimensional images of pure specimens of fish protamines (salmon, herring (clupeine, Y-l) and rainbow trout (Salmo irideus)). On the basis of these images, a model for the 3-dimensional structure of the fish protamines has been proposed (2).The known amino acid sequences of fish protamines show stretches of positively charged arginines, separated by regions of neutral amino acids (3). The proposed model for protamine structure (2) consists of an irregular, right-handed helix with the segments of adjacent arginines forming the loops of the coil.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

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