scholarly journals Bioenergetics of the Dictyostelium Kinesin-8 Motor Isoform

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 563
Author(s):  
Michael P. Koonce ◽  
Irina Tikhonenko
Keyword(s):  
Dictyostelium Discoideum ◽  
Functional Diversity ◽  
Functional Organization ◽  
Dynamic Properties ◽  
Biochemical Properties ◽  
Eukaryotic Cells ◽  
Accessory Proteins ◽  
Integral Role ◽  
Localization Data

The functional organization of microtubules in eukaryotic cells requires a combination of their inherent dynamic properties, interactions with motor machineries, and interactions with accessory proteins to affect growth, shrinkage, stability, and architecture. In most organisms, the Kinesin-8 family of motors play an integral role in these organizations, well known for their mitotic activities in microtubule (MT) length control and kinetochore interactions. In Dictyostelium discoideum, the function of Kinesin-8 remains elusive. We present here some biochemical properties and localization data that indicate that this motor (DdKif10) shares some motility properties with other Kinesin-8s but also illustrates differences in microtubule localization and depolymerase action that highlight functional diversity.

scholarly journals An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Yu Tang ◽  
Yuantai Wu ◽  
Sarah E. Herlihy ◽  
Francisco J. Brito-Aleman ◽  
Jose H. Ting ◽  
...  
Keyword(s):  
G Protein ◽  
Dictyostelium Discoideum ◽  
Protein A ◽  
G Protein Coupled Receptors ◽  
Eukaryotic Cells ◽  
Putative Receptor ◽  
Large Numbers ◽  
Eukaryotic Microbes ◽  
G Protein Coupled ◽  
Eukaryotic Microbe

ABSTRACT In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells (grlH¯/grlH OE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum. IMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion.

scholarly journals The proteasome: a macromolecular assembly designed for controlled proteolysis

1999 ◽  
Vol 354 (1389) ◽  
pp. 1501-1511 ◽  
Author(s):  
P. Zwickl ◽  
D. Voges ◽  
W. Baumeister
Keyword(s):  
Functional Organization ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Core Complex ◽  
Ubiquitin Pathway ◽  
Enzymatic Mechanism ◽  
Macromolecular Assembly ◽  
Regulatory Complex ◽  
Structure Assembly ◽  
Made In

In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome–ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel–shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them.

scholarly journals Sphingosine Kinase Regulates the Sensitivity of Dictyostelium discoideum Cells to the Anticancer Drug Cisplatin

2005 ◽  
Vol 4 (1) ◽  
pp. 178-189 ◽  
Author(s):  
Junxia Min ◽  
David Traynor ◽  
Andrew L. Stegner ◽  
Lei Zhang ◽  
Marie H. Hanigan ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Drug Targets ◽  
Sphingosine Kinase ◽  
Biochemical Properties ◽  
Sphingosine 1 Phosphate ◽  
Sphingosine Kinases ◽  
Cisplatin Sensitivity ◽  
Increased Sensitivity ◽  
Tumor Types ◽  
Null Mutants

ABSTRACT The drug cisplatin is widely used to treat a number of tumor types. However, resistance to the drug, which remains poorly understood, limits its usefulness. Previous work using Dictyostelium discoideum as a model for studying drug resistance showed that mutants lacking sphingosine-1-phosphate (S-1-P) lyase, the enzyme that degrades S-1-P, had increased resistance to cisplatin, whereas mutants overexpressing the enzyme were more sensitive to the drug. S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine kinase. We have identified two sphingosine kinase genes in D. discoideum—sgkA and sgkB—that are homologous to those of other species. The biochemical properties of the SgkA and SgkB enzymes suggest that they are the equivalent of the human Sphk1 and Sphk2 enzymes, respectively. Disruption of the kinases by homologous recombination (both single and double mutants) or overexpression of the sgkA gene resulted in altered growth rates and altered response to cisplatin. The null mutants showed increased sensitivity to cisplatin, whereas mutants overexpressing the sphingosine kinase resulted in increased resistance compared to the parental cells. The results indicate that both the SgkA and the SgkB enzymes function in regulating cisplatin sensitivity. The increase in sensitivity of the sphingosine kinase-null mutants was reversed by the addition of S-1-P, and the increased resistance of the sphingosine kinase overexpressor mutant was reversed by the inhibitor N,N-dimethylsphingosine. Parallel changes in sensitivity of the null mutants are seen with the platinum-based drug carboplatin but not with doxorubicin, 5-fluorouracil, and etoposide. This pattern of specificity is similar to that observed with the S-1-P lyase mutants and should be useful in designing therapeutic schemes involving more than one drug. This study identifies the sphingosine kinases as new drug targets for modulating the sensitivity to platinum-based drugs.

scholarly journals The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Open Biology ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 150008 ◽  
Author(s):  
Qiuhong Xiong ◽  
Can Ünal ◽  
Jan Matthias ◽  
Michael Steinert ◽  
Ludwig Eichinger
Keyword(s):  
Dictyostelium Discoideum ◽  
Legionella Pneumophila ◽  
Double Mutant ◽  
Eukaryotic Cells ◽  
Knock Out ◽  
Cellular Processes ◽  
Ubiquitinated Proteins ◽  
Complex Phenotypes ◽  
Independent Functions ◽  
Mutant Cells

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16 − and ATG9 − /16 − cells and compare them to the previously reported ATG9 − mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9 − and ATG16 − cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9 − /16 − double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9 − /16 − cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9 − /16 − cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.

scholarly journals Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
R Stefan Isaac ◽  
Fuguo Jiang ◽  
Jennifer A Doudna ◽  
Wendell A Lim ◽  
Geeta J Narlikar ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Surveillance System ◽  
Dynamic Properties ◽  
Eukaryotic Cells ◽  
Chromatin Remodelers ◽  
Nucleosomal Dna ◽  
Eukaryotic Chromatin ◽  
Versatile Tool

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

scholarly journals FANCD2-associated nuclease 1 partially compensates for the lack of Exonuclease 1 in mismatch repair.

2021 ◽  
Author(s):  
Katja Kratz ◽  
Mariela Artola-Borán ◽  
Saho Kobayashi-Era ◽  
Gene Koh ◽  
Goncalo Oliveira ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Familial Cancer ◽  
Germline Mutations ◽  
Biochemical Properties ◽  
Functional Complementation ◽  
Eukaryotic Cells ◽  
R Genes ◽  
Mutator Phenotype ◽  
Exonuclease 1

Germline mutations in the mismatch repair ( MM R) genes MSH2 , MSH6 , MLH1 and PMS2 are linked to cancer of the colon and other organs, characterised by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1 , encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes towards MMR in vivo : cells lacking both EXO1 and FAN1 have a MMR defect and display resistance to N -methyl- N -nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, implying that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

scholarly journals Single Molecule Light Field Microscopy

2020 ◽  
Author(s):  
Ruth R. Sims ◽  
Sohaib Abdul Rehman ◽  
Martin O. Lenz ◽  
Adam Clark ◽  
Edward W. Sanders ◽  
...  
Keyword(s):  
Single Molecule ◽  
Focal Plane ◽  
Light Field ◽  
Microlens Array ◽  
Diffraction Limit ◽  
Eukaryotic Cells ◽  
Depth Of Field ◽  
3D Localization ◽  
20 Nm ◽  
Localization Data

We introduce single molecule light field microscopy (SMLFM), a novel 3D single molecule localization technique that is capable of up to 20 nm isotropic precision across a 6 μm depth of field. SMLFM can be readily implemented by installing a refractive microlens array into the conjugate back focal plane of any widefield single molecule localization system. We demonstrate that 3D localization can be performed by post-processing 2D localization data generated by common, widely-used, algorithms. In this work we benchmark the performance of SMLFM and finally showcase its capabilities by imaging fluorescently labeled membranes of fixed eukaryotic cells below the diffraction limit.

scholarly journals Changes in Microbial Biomass, Activity, Functional Diversity, and Enzyme Activity in Tree Peony (Paeonia suffruticosa) Garden Soils

HortScience ◽  
2014 ◽  
Vol 49 (11) ◽  
pp. 1408-1413 ◽  
Author(s):  
Xiangdong Huang ◽  
Dong Xue ◽  
Lian Xue
Keyword(s):  
Microbial Biomass ◽  
Functional Diversity ◽  
Microbial Biomass Carbon ◽  
Soil Microbial Communities ◽  
Biochemical Properties ◽  
Paeonia Suffruticosa ◽  
Tree Peony ◽  
Growth Duration ◽  
Organic C ◽  
Soil Microbial

To understand the effects of tree peony (Paeonia suffruticosa) on soil microbiological and biochemical properties, soil samples were collected from tree peony growing sites with 3 growth years and four tree peony cultivars as well as from an adjacent wasteland in a tree peony garden at Luoyang, Henan Province of China. With the development of the tree peony garden ecosystem, soil microbial biomass carbon (Cmic), basal respiration (Rmic), Cmic as a percent of soil organic C (Cmic/Corg), and enzyme activities first increased and then decreased. For the tree peony cultivars Yao Huang and Dou Lu, Cmic, Rmic, Cmic/Corg, catalase, invertase, cellulose, proteinase, and phosphatase decreased after 5 years of growth, whereas urease decreased after 12 years. For the cultivars Er Qiao and Shou An Hong, catalase, proteinase, and phosphatase decreased after 5 years, whereas Cmic, Rmic, Cmic/Corg, invertase, cellulose, and urease decreased after 12 years. Biolog analysis indicated that the average well color development and microbial functional diversity were significantly greater at the 5-year sites than in the wasteland but decreased significantly as growth continued. The growth duration of tree peony had a greater effect on soil microbial communities than did tree peony cultivar.

scholarly journals Metabolic regulation of mitochondrial dynamics

2016 ◽  
Vol 212 (4) ◽  
pp. 379-387 ◽  
Author(s):  
Prashant Mishra ◽  
David C. Chan
Keyword(s):  
Adenosine Triphosphate ◽  
Metabolic Regulation ◽  
Dynamic Properties ◽  
Mitochondrial Dynamics ◽  
Cellular Signaling ◽  
Eukaryotic Cells ◽  
Dynamic Processes ◽  
Metabolic Function ◽  
Mitochondrial Population ◽  
Signaling Events

Mitochondria are renowned for their central bioenergetic role in eukaryotic cells, where they act as powerhouses to generate adenosine triphosphate from oxidation of nutrients. At the same time, these organelles are highly dynamic and undergo fusion, fission, transport, and degradation. Each of these dynamic processes is critical for maintaining a healthy mitochondrial population. Given the central metabolic function of mitochondria, it is not surprising that mitochondrial dynamics and bioenergetics reciprocally influence each other. We review the dynamic properties of mitochondria, with an emphasis on how these processes respond to cellular signaling events and how they affect metabolism.

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