Ralf Jansen | Eberhard Karls Universität Tübingen (original) (raw)

Papers by Ralf Jansen

Current Biology, 2003

cleared by centrifugation (5 min, 6,000 ϫ g) and were subsequently incubated for 30 min with CL-4... more cleared by centrifugation (5 min, 6,000 ϫ g) and were subsequently incubated for 30 min with CL-4B Sepharose. Monoclonal anti-myc Indirect Immunfluorescence Studies For immunoflourescence studies against myc-and HA-tagged pro-antibody 9E10 (Roche) was added to the extract and was incubated on ice for 1 hr following incubation with a mix of Protein G Sepharose teins, exponentially growing cells were fixed in formaldehyde (4% final concentration) for 60 min at the corresponding temperature, 4 Fast Flow (Pharmacia Biotech) and CL-4B Sepharose (1:2 ratio). Beads were washed once with 1ϫ BB including protease inhibitors pelleted, washed, and converted to spheroplasts. Spheroplasts were attached to poly-L lysine-coated slides and were sequentially and insulin and three times with 1ϫ BB. Proteins were released from beads with 50 l hot SDS-PAGE sample buffer. Myc epitope-incubated with mouse anti-myc (9E10, Roche) or rat anti-HA antibody (3F10, Roche), followed by either rabbit anti-mouse IgG or tagged myosins and HA epitope-tagged She4p were detected by Western blotting with antibodies 9E10 or 3F10 (rat anti-HA). goat anti-rat IgG coupled to Alexa-488 (both from Molecular Probes).

Current Biology, 2004

by amplifying DPM1 with primers 5Ј-TTTACTAGTCCGTGCTTAAGG GTTTCTA-3Ј and 5Ј-TTTGTCGACTAAAGACCAAATG... more by amplifying DPM1 with primers 5Ј-TTTACTAGTCCGTGCTTAAGG GTTTCTA-3Ј and 5Ј-TTTGTCGACTAAAGACCAAATGGTATAGC TGG-3Ј. The resulting product was cloned between the SpeI and Media and Yeast Strains XhoI sites of pPS1890. Media were prepared as described previously [S1]. Yeast transfor-

Current Opinion in Cell Biology, 2004

mRNA localization is a widespread post-transcriptional mechanism for targeting protein synthesis ... more mRNA localization is a widespread post-transcriptional mechanism for targeting protein synthesis to specific cellular sites. It is involved in the generation of cell polarity, asymmetric segregation of cell fate determinants and germ cell specification. Actin and microtubule filaments have key functions during RNA localization, especially during transport of mRNAs and anchoring at target sites. Recent advances in understanding the role of motors and filament systems have mainly resulted from the contribution of live imaging of mRNA movement and from the purification of putative localization ribonucleoproteins. There have also been new findings on the role of centrosomes in RNA localization.

Traffic, 2008

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of prot... more Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.

Proceedings of The National Academy of Sciences, 2007

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most pl... more Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargocomplex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargotranslocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosincargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.

Seminars in Cell & Developmental Biology, 2007

RNA localization is a widespread mechanism that allows cells to spatially control protein functio... more RNA localization is a widespread mechanism that allows cells to spatially control protein function by determining their sites of synthesis. In embryos, localized mRNAs are involved in morphogen gradient formation or the asymmetric distribution of cell fate determinants. In somatic cell types, mRNA localization contributes to local assembly of protein complexes or facilitates protein targeting to organelles. Long-distance transport of specific mRNAs in plants allows coordination of developmental processes between different plant organs. In this review, we will discuss the biological significance of different patterns of mRNA localization.

Current Biology, 2006

Plasmid Constructions Plasmid pRJ741 (pG14-prGPD1-MS2CP-RedStar) was constructed by ligating pG14... more Plasmid Constructions Plasmid pRJ741 (pG14-prGPD1-MS2CP-RedStar) was constructed by ligating pG14-GPD1-MS2CP-GFP [S1], which was digested with BamHI and SalI to release GFP, with a NLS-HA-MS2CP fragment (released by BamHI and NotI from plasmid pG14-GPD1-MS2CP-GFP) and the coding sequence of the RedStar fluorescence protein amplified with primers RPO1305 (5 0 -GGCAATGGGCGGCCGCTGG AGCTGGAGCTGGTGCAGG-3 0 ) and RPO1306 (5 0 -GGCCGTCGACTT ACAAGAACAAGTGGTGTCTAC-3 0 ). The primers contain a NotI or SalI site (underlined) used for cloning. In the resulting plasmid, Red-Star has replaced GFP.

Current Biology, 2003

cleared by centrifugation (5 min, 6,000 ϫ g) and were subsequently incubated for 30 min with CL-4... more cleared by centrifugation (5 min, 6,000 ϫ g) and were subsequently incubated for 30 min with CL-4B Sepharose. Monoclonal anti-myc Indirect Immunfluorescence Studies For immunoflourescence studies against myc-and HA-tagged pro-antibody 9E10 (Roche) was added to the extract and was incubated on ice for 1 hr following incubation with a mix of Protein G Sepharose teins, exponentially growing cells were fixed in formaldehyde (4% final concentration) for 60 min at the corresponding temperature, 4 Fast Flow (Pharmacia Biotech) and CL-4B Sepharose (1:2 ratio). Beads were washed once with 1ϫ BB including protease inhibitors pelleted, washed, and converted to spheroplasts. Spheroplasts were attached to poly-L lysine-coated slides and were sequentially and insulin and three times with 1ϫ BB. Proteins were released from beads with 50 l hot SDS-PAGE sample buffer. Myc epitope-incubated with mouse anti-myc (9E10, Roche) or rat anti-HA antibody (3F10, Roche), followed by either rabbit anti-mouse IgG or tagged myosins and HA epitope-tagged She4p were detected by Western blotting with antibodies 9E10 or 3F10 (rat anti-HA). goat anti-rat IgG coupled to Alexa-488 (both from Molecular Probes).

Current Biology, 2004

by amplifying DPM1 with primers 5Ј-TTTACTAGTCCGTGCTTAAGG GTTTCTA-3Ј and 5Ј-TTTGTCGACTAAAGACCAAATG... more by amplifying DPM1 with primers 5Ј-TTTACTAGTCCGTGCTTAAGG GTTTCTA-3Ј and 5Ј-TTTGTCGACTAAAGACCAAATGGTATAGC TGG-3Ј. The resulting product was cloned between the SpeI and Media and Yeast Strains XhoI sites of pPS1890. Media were prepared as described previously [S1]. Yeast transfor-

Current Opinion in Cell Biology, 2004

mRNA localization is a widespread post-transcriptional mechanism for targeting protein synthesis ... more mRNA localization is a widespread post-transcriptional mechanism for targeting protein synthesis to specific cellular sites. It is involved in the generation of cell polarity, asymmetric segregation of cell fate determinants and germ cell specification. Actin and microtubule filaments have key functions during RNA localization, especially during transport of mRNAs and anchoring at target sites. Recent advances in understanding the role of motors and filament systems have mainly resulted from the contribution of live imaging of mRNA movement and from the purification of putative localization ribonucleoproteins. There have also been new findings on the role of centrosomes in RNA localization.

Traffic, 2008

Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of prot... more Intracellular mRNA localization is a common mechanism to achieve asymmetric distributions of proteins. Previous studies have revealed that in a number of cell types, different mRNA species are localized by the same transport machinery. However, it has been unclear if these individual mRNA species are specifically sorted into separate or common ribonucleoprotein (RNP) particles before or during transport. Using budding yeast as a model system, we analyzed the intracellular movement of individual pairs of localized mRNA in live cells. Yeast cells localize more than 20 different mRNAs to the bud with the help of the Myo4p/She3p/She2p protein complex. For live cell imaging, mRNA pairs were tagged with tandem repeats of either bacteriophage MS2 or lambda boxB RNA sequences and fluorescently labeled by fusion protein constructs that bind to the RNA tag sequences. Using three-dimensional, single-particle tracking with dual-color detection, we have tracked the transport of two different localized mRNA species in real time. Our observations show that different localized mRNAs are coassembled into common RNP particles and cotransported in a directional manner to the target site. Nonlocalized mRNAs or mutant mRNAs that lack functional localization signals form separate particles that are not transported to the bud. This study reveals a high degree of co-ordination of mRNA trafficking in budding yeast.

Proceedings of The National Academy of Sciences, 2007

Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most pl... more Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargocomplex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargotranslocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosincargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport.

Seminars in Cell & Developmental Biology, 2007

RNA localization is a widespread mechanism that allows cells to spatially control protein functio... more RNA localization is a widespread mechanism that allows cells to spatially control protein function by determining their sites of synthesis. In embryos, localized mRNAs are involved in morphogen gradient formation or the asymmetric distribution of cell fate determinants. In somatic cell types, mRNA localization contributes to local assembly of protein complexes or facilitates protein targeting to organelles. Long-distance transport of specific mRNAs in plants allows coordination of developmental processes between different plant organs. In this review, we will discuss the biological significance of different patterns of mRNA localization.

Current Biology, 2006

Plasmid Constructions Plasmid pRJ741 (pG14-prGPD1-MS2CP-RedStar) was constructed by ligating pG14... more Plasmid Constructions Plasmid pRJ741 (pG14-prGPD1-MS2CP-RedStar) was constructed by ligating pG14-GPD1-MS2CP-GFP [S1], which was digested with BamHI and SalI to release GFP, with a NLS-HA-MS2CP fragment (released by BamHI and NotI from plasmid pG14-GPD1-MS2CP-GFP) and the coding sequence of the RedStar fluorescence protein amplified with primers RPO1305 (5 0 -GGCAATGGGCGGCCGCTGG AGCTGGAGCTGGTGCAGG-3 0 ) and RPO1306 (5 0 -GGCCGTCGACTT ACAAGAACAAGTGGTGTCTAC-3 0 ). The primers contain a NotI or SalI site (underlined) used for cloning. In the resulting plasmid, Red-Star has replaced GFP.