EB1 and EB3 Control CLIP Dissociation from the Ends of Growing Microtubules

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.

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Mitophagy as a stress response in mammalian cells and in respiring S. cerevisiae

Biochemical Society Transactions ◽

10.1042/bst20150278 ◽

2016 ◽

Vol 44 (2) ◽

pp. 541-545 ◽

Cited By ~ 7

Author(s):

Hagai Abeliovich ◽

Jörn Dengjel

Keyword(s):

Mammalian Cells ◽

Late Onset ◽

Specific Form ◽

Mitochondrial Quality Control ◽

Evolutionarily Conserved ◽

Autophagic Degradation ◽

Relationship Of ◽

To Receive ◽

The Relationship ◽

Significant Attention

The degradation of malfunctioning or superfluous mitochondria in the lysosome/vacuole is an important housekeeping function in respiring eukaryotic cells. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy, and plays a role in physiological homoeostasis as well as in the progression of late-onset diseases. Although the mechanism of bulk degradation by macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. In this mini-review, we introduce mitophagy as a form of mitochondrial quality control and proceed to provide specific examples from yeast and mammalian systems. We then discuss the relationship of mitophagy to mitochondrial stress, and provide a broad mechanistic overview of the process with an emphasis on evolutionarily conserved pathways.

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RNA Interference Screening Identifies Novel Roles for RhoBTB1 and RhoBTB3 in Membrane Trafficking Events in Mammalian Cells

Cells ◽

10.3390/cells9051089 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1089 ◽

Cited By ~ 1

Author(s):

Maeve Long ◽

Tilen Kranjc ◽

Margaritha M. Mysior ◽

Jeremy C. Simpson

Keyword(s):

Rna Interference ◽

Golgi Complex ◽

Membrane Trafficking ◽

Mammalian Cells ◽

Rho Gtpase ◽

Family Members ◽

Membrane Traffic ◽

Small Gtpases ◽

Endomembrane System ◽

Rho Family

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

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Genetic and Functional Interaction of Evolutionarily Conserved Regions of the Prp18 Protein and the U5 snRNA

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2107-2116.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2107-2116 ◽

Cited By ~ 9

Author(s):

Dagmar Bačíková ◽

David S. Horowitz

Keyword(s):

Functional Relationship ◽

Mrna Splicing ◽

Second Step ◽

Functional Interaction ◽

Synthetic Lethal ◽

Conserved Regions ◽

Evolutionarily Conserved ◽

Conserved Region ◽

Relationship Of ◽

U5 Snrna

ABSTRACT Both the Prp18 protein and the U5 snRNA function in the second step of pre-mRNA splicing. We identified suppressors of mutant prp18 alleles in the gene for the U5 snRNA (SNR7). The suppressors' U5 snRNAs have either a U4-to-A or an A8-to-C mutation in the evolutionarily invariant loop 1 of U5. Suppression is specific for prp18 alleles that encode proteins with mutations in a highly conserved region of Prp18 which forms an unstructured loop in crystals of Prp18. The snr7 suppressors partly restored the pre-mRNA splicing activity that was lost in the prp18 mutants. The close functional relationship of Prp18 and U5 is emphasized by the finding that two snr7 alleles, U5A and U6A, are dominant synthetic lethal with prp18 alleles. Our results support the idea that Prp18 and the U5 snRNA act in concert during the second step of pre-mRNA splicing and suggest a model in which the conserved loop of Prp18 acts to stabilize the interaction of loop 1 of the U5 snRNA with the splicing intermediates.

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The Evolutionary Conserved Transmembrane BAX Inhibitor Motif (TMBIM) Containing Protein Family Members 5 and 6 Are Essential for the Development and Survival of Drosophila melanogaster

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.666484 ◽

2021 ◽

Vol 9 ◽

Author(s):

Li Zhang ◽

Sebastian Buhr ◽

Aaron Voigt ◽

Axel Methner

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Developmental Stages ◽

Family Members ◽

Protein Family ◽

Expression Levels ◽

Atp Production ◽

Evolutionarily Conserved ◽

Human Proteins ◽

Er Calcium

The mammalian Transmembrane BAX Inhibitor Motif (TMBIM) protein family consists of six evolutionarily conserved hydrophobic proteins that affect programmed cell death and the regulation of intracellular calcium levels. The bacterial ortholog BsYetJ is a pH-dependent calcium channel. We here identified seven TMBIM family members in Drosophila melanogaster and describe their expression levels in diverse tissues and developmental stages. A phylogenetic analysis revealed that CG30379 represents the ortholog of human TMBIM4 although these two proteins are much less related than TMBIM5 (CG2076 and CG1287/Mics1) and TMBIM6 (CG7188/Bi-1) to their respective orthologs. For TMBIM1-3 the assignment is more dubious because the fly and the human proteins cluster together. We conducted a functional analysis based on expression levels and the availability of RNAi lines. This revealed that the ubiquitous knockdown of CG3798/Nmda1 and CG3814/Lfg had no effect on development while knockdown of CG2076/dTmbim5 resulted in death at the pupa stage and knockdown of CG7188/dTmbim6 in death at the embryonic stage. Ubiquitous knockdown of the second TMBIM5 paralog CG1287/Mics1 ensued in male sterility. Knockdown of dTmbim5 and 6 in muscle and neural tissue also greatly reduced lifespan through different mechanisms. Knockdown of the mitochondrial family member dTmbim5 resulted in reduced ATP production and a pro-apoptotic expression profile while knockdown of the ER protein dTmbim6 increased the ER calcium levels similar to findings in mammalian cells. Our data demonstrate that dTmbim5 and 6 are essential for fly development and survival but affect cell survival through different mechanisms.

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Apoptosis and development

Essays in Biochemistry ◽

10.1042/bse0390011 ◽

2003 ◽

Vol 39 ◽

pp. 11-24 ◽

Cited By ~ 6

Author(s):

Justin V McCarthy

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Cell Number ◽

Model Organisms ◽

Biological Processes ◽

Nematode Caenorhabditis Elegans ◽

Apoptotic Pathway ◽

Genetic Pathways ◽

Evolutionarily Conserved ◽

Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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Functional Relationship of Polymerization Sites of Vertebrate Fibrinogens

10.1055/s-0038-1665661 ◽

1979 ◽

Author(s):

C Cierniewski ◽

T Krajewski ◽

E Janiak

Keyword(s):

Gel Electrophoresis ◽

Functional Relationship ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Functional Similarity ◽

Fractionation Method ◽

Relationship Of ◽

Fibrin Monomers ◽

Fibrinogen Molecule ◽

Cold Ethanol Fractionation

Various studies on the interaction of immobilized mammalian fibrinogen and fibrin monomers with some fibrinogen derivatives demonstrated the presence of two sets of polymerization sites in the mammalian fibrinogen molecule. We obtained the same results while investigating the fibrinogen molecules of other classes of vertebrates /Pisces. Amphibia. Aves/. Despite significant differences among their subunit structures, all of them contain polymerization sites homologous to mammalian counterparts. Moreover, due to great functional similarity, fibrinogen or fibrin monomers of the analyzed species of Pisces. Amphibia. Aves and Mammalia interacted in a specific way with immobilized pig fibrin monomers or fibrinogen, respectively. Using these pig affinity adsorbents, fibrinogen and fibrin monomers of different vertebrates were isolated directly from plasma and analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide compositions of eluted proteins were identical to those obtained for corresponding fibrinogen preparations isolated by cold-ethanol fractionation method. It appears to indicate that the nature of polymerization sites in vertebrate fibrinogens is alike.

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