Plasmodesmata-like intercellular connections by plant remorin in animal cells

SummaryPlasmodesmata (PD) are crucial structures for intercellular communication in multicellular plants with remorins being their crucial plant-specific structural and functional constituents. The PD biogenesis is an intriguing but poorly understood process. By expressing an Arabidopsis remorin protein in mammalian cells, we have reconstituted a PD-like filamentous structure, termed remorin filament (RF), connecting neighboring cells physically and physiologically. Notably, RFs are capable of transporting macromolecules intercellularly, in a way similar to plant PD. With further super-resolution microscopic analysis and biochemical characterization, we found that RFs are also composed of actin filaments, forming the core skeleton structure, aligned with the remorin protein. This unique heterologous filamentous structure might explain the molecular mechanism for remorin function as well as PD construction. Furthermore, remorin protein exhibits a specific distribution manner in the plasma membrane in mammalian cells, representing a lipid nanodomain, depending on its lipid modification status. Our studies not only provide crucial insights into the mechanism of PD biogenesis, but also uncovers unsuspected fundamental mechanistic and evolutionary links between intercellular communication systems of plants and animals.SignificanceRemorin is rising as a crucial lipid microdomain marker, as well as an essential component of plasmodesmata in plants. However, the biological role of remorin in plants is elusive. With a heterologous system, we found that remorin expression is able to form intercellular filamentous structure, namely remorin filament (RF), connecting neighboring mammalian cells functionally. By employing multiple approaches, we tested the functionality of RFs, as well as investigated their structures. RFs highly resemble plant plasmodesmata, in many ways, such as its morphology, molecular constitutes, and its ability to transport macromolecules intercellularly. Our study provides novel insights into the biogenesis of plasmodesmata and uncovers fundamental evolutionary links in molecular construction of intercellular connections in both plants and animals.

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Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.325 ◽

1997 ◽

Vol 8 (2) ◽

pp. 325-339 ◽

Cited By ~ 119

Author(s):

J M Jürgensmeier ◽

S Krajewski ◽

R C Armstrong ◽

G M Wilson ◽

T Oltersdorf ◽

...

Keyword(s):

Cell Death ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Mammalian Cells ◽

Animal Cell ◽

Chromatin Condensation ◽

Interleukin 1 ◽

Microscopic Analysis ◽

Animal Cells ◽

Electron Microscopic

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031391 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1391

Author(s):

Andrey Kropotov ◽

Veronika Kulikova ◽

Kirill Nerinovski ◽

Alexander Yakimov ◽

Maria Svetlova ◽

...

Keyword(s):

Nicotinic Acid ◽

Mammalian Cells ◽

Experimental Models ◽

The Body ◽

Nucleoside Transporters ◽

Hek293 Cells ◽

Nucleoside Transporter ◽

Vitamin B3 ◽

Animal Cells ◽

Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

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Biochemical characterization of the golgi complex of mammalian cells

Journal of Supramolecular Structure ◽

10.1002/jss.400020517 ◽

1974 ◽

Vol 2 (5-6) ◽

pp. 737-750 ◽

Cited By ~ 23

Author(s):

Becca Fleischer ◽

Fernando Zambrano ◽

Sidney Fleischer

Keyword(s):

Golgi Complex ◽

Mammalian Cells ◽

Biochemical Characterization

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Adhesion-dependent and Contractile Ring-independent Equatorial Furrowing during Cytokinesis in Mammalian Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e05-03-0233 ◽

2005 ◽

Vol 16 (8) ◽

pp. 3865-3872 ◽

Cited By ~ 67

Author(s):

Masamitsu Kanada ◽

Akira Nagasaki ◽

Taro Q.P. Uyeda

Keyword(s):

Mammalian Cells ◽

Rat Kidney ◽

Myosin Ii ◽

Rho Kinase ◽

Cellular Slime Mold ◽

Animal Cells ◽

Contractile Ring ◽

Normal Rat ◽

Cellular Slime ◽

Coated Surfaces

Myosin II-dependent contraction of the contractile ring drives equatorial furrowing during cytokinesis in animal cells. Nonetheless, myosin II-null cells of the cellular slime mold Dictyostelium divide efficiently when adhering to substrates by making use of polar traction forces. Here, we show that in the presence of 30 μM blebbistatin, a potent myosin II inhibitor, normal rat kidney (NRK) cells adhering to fibronectin-coated surfaces formed equatorial furrows and divided in a manner strikingly similar to myosin II-null Dictyostelium cells. Such blebbistatin-resistant cytokinesis was absent in partially detached NRK cells and was disrupted in adherent cells if the advance of their polar lamellipodia was disturbed by neighboring cells. Y-27632 (40 μM), which inhibits Rho-kinase, was similar to 30 μM blebbistatin in that it inhibited cytokinesis of partially detached NRK cells but only prolonged furrow ingression in attached cells. In the presence of 100 μM blebbistatin, most NRK cells that initiated anaphase formed tight furrows, although scission never occurred. Adherent HT1080 fibrosarcoma cells also formed equatorial furrows efficiently in the presence of 100 μM blebbistatin. These results provide direct evidence for adhesion-dependent, contractile ring-independent equatorial furrowing in mammalian cells and demonstrate the importance of substrate adhesion for cytokinesis.

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Design of a Retrovirus-Derived Vector for Expression and Transduction of Exogenous Genes in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.3.6.1123-1132.1983 ◽

1983 ◽

Vol 3 (6) ◽

pp. 1123-1132

Author(s):

Archibald S. Perkins ◽

Paul T. Kirschmeier ◽

Sebastiano Gattoni-Celli ◽

I. Bernard Weinstein

Keyword(s):

Mammalian Cells ◽

High Efficiency ◽

Leukemia Virus ◽

Restriction Enzymes ◽

Virus Genome ◽

Opposite Polarity ◽

High Yield ◽

Animal Cells ◽

Murine Sarcoma Virus ◽

Sarcoma Virus

We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5′ and 3′ LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique Pst I site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt + colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5′ LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3′ LTR gave an 80% reduction in activity. Thus, both 5′ and 3′ LTR sequences are essential for optimal gpt expression, although the 5′ LTR appears to play a more important role. When the LTR- gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt + phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.

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The effects of D- and L-threo-chloramphenicol on the early development of the chick embryo

Development ◽

10.1242/dev.13.3.341 ◽

1965 ◽

Vol 13 (3) ◽

pp. 341-356

Author(s):

F. S. Billett ◽

Rosalba Collini ◽

Louie Hamilton

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Chick Embryo ◽

Messenger Rna ◽

Mammalian Cells ◽

Animal Cells ◽

Inhibit Protein Synthesis ◽

Acid Complex ◽

Amino Acid Complex ◽

Bacterial Systems

In many bacterial systems chloramphenicol has been shown to inhibit protein synthesis (Hahn & Wisseman, 1951; Gale & Folkes, 1953). The precise mechanism of this inhibition is not clear, although the evidence suggests that the interaction of the soluble RNA-amino acid complex with the ribosomes is prevented because the attachment of the messenger RNA to the ribosomes is itself impaired (Lacks & Gros, 1959; Nathans & Lipman, 1961; Jardetsky & Julian, 1964; Julian & Jardetsky, 1964). In contrast to its effect on bacterial systems, chloramphenicol has been reported to have little or no action on the protein synthesis by cell-free extracts of mammalian cells (Rendi, 1959; Ehrenstein & Lipmann, 1961). A basis for this resistance has been proposed by Vazquez (1964), who finds that whereas bacterial ribosomes bind chloramphenicol, ribosomes from other organisms do not. Nevertheless, it cannot be stated with any confidence that chloramphenicol has no effect on the protein synthesis of animal cells.

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Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.2080-2089.1985 ◽

1985 ◽

Vol 5 (8) ◽

pp. 2080-2089

Author(s):

C T Wake ◽

F Vernaleone ◽

J H Wilson

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Animal Cells ◽

Linear Molecules ◽

Sv40 Genome ◽

Foreign Dna ◽

The Individual ◽

Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

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Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.2080 ◽

1985 ◽

Vol 5 (8) ◽

pp. 2080-2089 ◽

Cited By ~ 47

Author(s):

C T Wake ◽

F Vernaleone ◽

J H Wilson

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Animal Cells ◽

Linear Molecules ◽

Sv40 Genome ◽

Foreign Dna ◽

The Individual ◽

Sv40 Dna

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Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽

10.3390/cells8020162 ◽

2019 ◽

Vol 8 (2) ◽

pp. 162

Author(s):

Marianne Grafe ◽

Petros Batsios ◽

Irene Meyer ◽

Daria Lisin ◽

Otto Baumann ◽

...

Keyword(s):

Electron Microscopy ◽

Nuclear Envelope ◽

Model Organism ◽

Super Resolution ◽

Nuclear Lamina ◽

Stimulated Emission ◽

Structural Level ◽

Animal Cells ◽

Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

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