Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants.

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation, and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

Simultaneous imaging of Pseudomonas fluorescens WCS365 populations expressing three different autofluorescent proteins in the rhizosphere: new perspectives for studying microbial communities.

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

B-type granule containing protrusions and interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression.

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch granules in wheat endosperm cells was studied by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) after expression and targeting of fluorescent protein into amyloplasts. Both techniques demonstrated the presence of protrusions emanating from A-type granules-containing amyloplasts and the presence of B-type starch granules in these evaginations. Moreover, CLSM recordings demonstrated the interconnection of the amyloplasts by these protrusions, suggesting a possible role of these protrusions in interplastid communication.

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum.

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).

Interactions between coiled-coil proteins: Drosophila lamin Dm0 binds to the bicaudal-D protein.

In a yeast two-hybrid screen we identified an interaction between Drosophila lamin Dm0, a structural nuclear protein, and BICD, a protein involved in oocyte development. The interaction can be reconstituted in vitro and takes place between segments of both proteins predicted to form coiled coils. The affinity for lamin Dm0 of the minimal binding site on BICD is modulated in a complex fashion by other BICD segments. A point mutation, F684I, that causes the dominant, bicaudal, Bic-D phenotype inhibits lamin binding in the context of the minimal lamin-binding site, but not in a larger BICD fragment. The minimal lamin-binding site of BICD binds to a few other coiled-coil proteins, but binding to these proteins is not influenced by the F684I point mutation, suggesting that the interaction with lamin may play a role in Bic-D function. Our structural studies demonstrated that BICD is 60-70% alpha-helical, is a dimer, and consists of two parts: a thin rod-shaped part of about 32 nm, and a thicker rod-shaped part of about 26 nm. Likely, the thinner rod-shaped part of full-length BICD consists of the N-terminal half of the protein, and the lamin-binding site is located within the thicker rod-shaped part.

Ectopic overexpression of Drosophila lamin C is stage-specific lethal.

To gain insight into the function of the developmentally regulated A-type lamins we transformed Drosophila melanogaster with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C was expressed ectopically after heat shock of embryos and localized to the nucleus. No phenotypic change was observed after lamin C expression in embryos that normally do not contain lamin C. However, ectopic expression of lamin C during most larval (but not pupal) stages stalled growth, inhibited ecdysteroid signaling (in particular during the larval-prepupal transition), resulted in development of melanotic tumors, and finally caused death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity.

Phosphorylation of the major Drosophila lamin in vivo: site identification during both M-phase (meiosis) and interphase by electrospray ionization tandem mass spectrometry.

Phosphorylation can have profound effects on the properties of nuclear lamins. For instance, phosphorylation of specific sites on mammalian lamins drastically alters their propensity to polymerize. Relatively little is known about the effects of phosphorylation during interphase and about phosphorylation of invertebrate nuclear lamins. Here, using electrospray ionization tandem mass spectrometry, we determined the phosphorylation sites of both interphase and M-phase isoforms of nuclear lamin Dm from Drosophila melanogaster. Interphase lamins are phosphorylated at three sites: two of these sites (Ser25 and a site located between residues 430 and 438) flank the alpha-helical rod domain, whereas the third site (Ser595) is located close to the C-terminus. The M-phase lamin isoform is phosphorylated predominantly at Ser45, a residue contained within a sequence matching the consensus site for phosphorylation by cdc2 kinase. Our study confirms the important role in vivo for cdc2 kinase in M-phase disassembly of nuclear lamins and provides the basis for understanding Drosophila lamin phosphorylation during interphase.

A tailless Drosophila lamin Dm0 fragment reveals lateral associations of dimers.

An early step in nuclear lamin polymerization is the longitudinal, head-to-tail association of lamin dimers through the highly conserved end segments of their rod domains. Lamin fragments lacking the carboxy-terminal tail domain (tailless lamins) form long, thin protofilaments more readily than full-length lamins do. By morphology alone it cannot be ascertained whether tailless lamin protofilaments also arise through head-to-tail association of dimers. Here, we studied by transmission electron microscopy which types of interaction are important for formation of protofilaments by a tailless lamin fragment derived from Drosophila lamin Dm0. First, we measured the lengths of tailless lamin filaments shorter than 200 nm. Many particles were longer than one and shorter than two dimers. Second, we labeled filaments formed by tailless lamin with a Fab fragment derived from a monoclonal antibody binding to an epitope residing in the amino-terminal head domain. The pattern of Fab fragments bound to lamin filaments clearly showed that most tailless lamin dimers are not arranged in a head-to-tail fashion. Third, we mutated tailless lamin using two point mutations known to inhibit head-to-tail association of full-length lamin. Thus mutated tailless lamin still formed filaments. We interpret these data according to models involving lateral associations between dimers of cytoplasmic intermediate filament proteins, using the lengths of short tailless lamin filaments as constraints. The data also demonstrate that a segment of at most 41 amino acids of the lamin tail domain is sufficient to bring about the change of mainly lateral to mainly longitudinal assembly.

Nuclear lamins: their structure, assembly, and interactions.

Nuclear lamins are intermediate filament-type proteins that are the major building blocks of the nuclear lamina, a fibrous proteinaceous meshwork underlying the inner nuclear membrane. Lamins can also be localized in the nuclear interior, in a diffuse or spotted pattern. Nuclei assembled in vitro in the absence of lamins are fragile, indicating that lamins mechanically stabilize the cell nucleus. Available evidence also indicates a role for lamins in DNA replication, chromatin organization, spatial arrangement of nuclear pore complexes, nuclear growth, and anchorage of nuclear envelope proteins. In this review we summarize the current state of knowledge on the structure, assembly, and possible functional roles of nuclear lamins, emphasizing the information concerning the ability of nuclear lamins to self-assemble into distinct oligomers and polymers.

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA.

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

In vivo association of lamins with nucleic acids in Drosophila melanogaster.

A 32P-labeling strategy was developed to study the interaction(s) in tissue culture cells between proteins and nucleic acids. Interphase and mitotic nuclear lamins were studied in Drosophila Kc cells. After bromodeoxyuridine incorporation and in vivo photo-crosslinking with 366 nm light, it was found that interphase lamins were associated with nucleic acid. Interactions with DNA as well as RNA were detected. In contrast, interaction of nucleic acids with mitotic lamin was not observed. Photo-crosslinking in the presence of antibiotics distamycin and/or chromomycin suggested that interphase lamins interacted with both A-T-rich DNA and G-C-rich DNA; interactions with G-C-rich DNA predominated. These results have implications for understanding the interphase organization of the higher eukaryotic cell nucleus as well as the transition of cells from interphase to mitosis. A model of nuclear organization, consistent with our results, is proposed.

Assembly of Drosophila lamin Dm0 and C mutant proteins studied with the baculovirus system.

Despite extensive knowledge of the in vitro polymerization properties of nuclear lamins, it is still not well understood how the nuclear lamina assembles in vivo. To learn more about the relationship between in vitro and in vivo polymerization of nuclear lamins, we expressed Drosophila lamin Dm0, mutant proteins, having well defined alterations of their in vitro polymerization properties, in Sf9 cells using the baculovirus system. All lamin Dm0 mutants assembled into fibrillar aggregates indistinguishable in morphology from those assembled by the wild-type protein. However, in contrast to wild-type lamin Dm0, mutant proteins were extracted with buffers of physiological ionic strength and pH containing Triton X-100. These results indicate that various types of lamin dimer-dimer interactions can be disrupted without affecting the morphology of the lamin Dm0 polymer. However, all types of dimer-dimer interactions tested appear to be important for full polymer stability. In addition, we analyzed the polymer formation of two Drosophila lamin C mutants and found that a segment in the carboxy-terminal tail domain is required for assembly of lamin C paracrystals at the nuclear lamina.

Identification of a conserved phosphorylation site modulating nuclear lamin polymerization.

Mitotic lamin disassembly results from phosphorylation at specific sites. In vitro, lamins can form head-to-tail polymers that disassemble upon phosphorylation by cdc2 kinase. A co-immunoprecipitation assay, employing Drosophila nuclear lamin Dm0 fragments was used to study the effect of phosphorylation on head-to-tail binding. Phosphorylation of serine-50 by cAMP-dependent kinase inhibited head-to-tail binding in the same manner as phosphorylation of serine-42 by cdc2 kinase. Results suggest that multiple pathways may be employed to disassemble nuclear lamins in vivo.

In vitro assembly of Drosophila lamin Dm0--lamin polymerization properties are conserved.

Vertebrate nuclear lamins exhibit polymerization properties that are remarkably different from those of vertebrate cytoplasmic intermediate filament (IF) proteins. Notably, under conditions where vertebrate cytoplasmic IF proteins form tetramers consisting of laterally associated dimers, nuclear lamin dimers associate longitudinally into head-to-tail polymers. Also, in vitro, nuclear lamins readily form paracrystalline fibers, rather than stable 10-nm filaments. To investigate whether these properties are also shared with invertebrate nuclear lamins, we analyzed in considerable detail the polymerization behavior of recombinant full-length lamin Dm0 from the invertebrate Drosophila melanogaster. This lamin differs substantially from vertebrate lamins in its primary structure. We also analyzed lamin Dm0-derived fragments lacking either the head domain (headless), the tail domain (tailless), or both (rod). Like vertebrate lamins, full-length Drosophila lamin Dm0 assembled into head-to-tail polymers, with little or no formation of tetramers by lateral association of dimers. This longitudinal assembly was severely inhibited by deletion of the head domain. Removal of the tail domain led to increased formation of filamentous polymers. Under appropriate conditions, full-length Drosophila lamin Dm0 as well as the three lamin Dm0-derived fragments assembled into paracrystalline fibers. No steady-state condition tested yielded assembly of 10-nm filaments resembling those formed by vertebrate cytoplasmic IF proteins. These findings indicate that the in vitro assembly behavior of nuclear lamins is highly conserved but distinct from that of cytoplasmic IF proteins, thus evidencing its functional importance.

Assembly of A- and B-type lamins studied in vivo with the baculovirus system.

We have expressed an A-type lamin (Xenopus lamin A), a probable A-type lamin (Drosophila lamin C), two B-type lamins (Xenopus lamin LI, Drosophila lamin Dmo), and two mutants of Xenopus lamin A in Sf9 cells. All proteins were synthesized at high levels resulting in formation of paracrystals with an axial repeat of 18.5-20.0 nm by A-type lamins; in contrast B-type lamins assembled into aggregates with a fibrillar ultrastructure. Of the four wild-type proteins analyzed only lamin Dmo was found in the nuclear compartment of Sf9 cells in association with the lamina whereas the three other lamins assembled into polymers localized in the cytoplasm as well as the nucleoplasm. The Xenopus lamin A mutant lacking the complete carboxy-terminal tail assembled in the cytoplasm into long filament bundles consisting of fibrils of less than 6 nm diameter. In vitro the non-helical amino-terminal head domain of lamins is required for the formation of 'head-to-tail' polymers. A lamin A mutant lacking this domain could be efficiently extracted from Sf9 cells with physiological buffers containing Triton X-100, demonstrating the importance of this domain for lamin assembly in vivo.

Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding.

Polymerization of intermediate filament proteins results from interactions among several distinct binding sites on the constituent proteins. Nuclear lamin head-to-tail polymers arise from one such interaction. We studied this binding using Drosophila lamin Dm0-derived fragments containing either the NH2-terminal or COOH-terminal binding site with a combination of co-immunoprecipitation, yeast two-hybrid, analytical ultracentrifugation, and electron microscopic assays. Fragment binding and full-length lamin head-to-tail polymerization were similar to each other in morphology, buffer requirements, and inhibition after phosphorylation with cdc2 kinase. Deletion analysis localized the binding sites to the ends of the rod domain that are highly conserved among all intermediate filament proteins. Point mutants, defective in binding, were isolated. Two were identical to point mutations in specific human keratin genes known to affect keratin assembly and to cause genetic skin diseases. Results further indicate that the binding sites only function in specific sequence contexts and that binding can be modulated by elements outside the binding sites (like the cdc2 kinase phosphorylation site). Our data indicate that one type of interaction in intermediate filament protein polymerization is the longitudinal binding of dimers via the conserved end segments of the coiled-coil rod domain.

DNA from Drosophila melanogaster beta-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ.

A DNA fragment designated lambda 20p1.4 binds in vitro to polymerized Drosophila melanogaster lamin. In situ hybridization of lambda 20p1.4 to isolated polytene chromosomes revealed localization at the chromocenter and to the 49 CD region on the right arm of chromosome 2. About 120 copies of sequences homologous to lambda 20p1.4 were detected per haploid genome. Nucleotide (nt) sequence analysis demonstrates that lambda 20p1.4 is an A + T-rich, 1327-bp fragment containing four repeated units between nt 595 and 919. Results suggest that lamin interacts with a region of lambda 20p1.4 between nt 300 and 1000. Confocal immunofluorescence co-localization demonstrates that in situ, the major locus of lambda 20p1.4 hybridization, the chromocenter, is found juxtaposed to the nuclear envelope (lamina). This is the first demonstration that a DNA sequence that binds specifically to nuclear lamins in vitro, is located at or near the nuclear envelope in situ and, presumably, in vivo.

Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state.

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized alpha-helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.

Expression of Drosophila lamin C is developmentally regulated: analogies with vertebrate A-type lamins.

Vertebrate nuclear lamins form a multigene family with developmentally controlled expression. In contrast, invertebrates have long been thought to contain only a single lamin, which in Drosophila is the well-characterized lamin Dm0. Recently, however, a Drosophila cDNA clone (pG-IF) has been identified that codes for an intermediate filament protein which harbors a nuclear localization signal but lacks a carboxy-terminal CAAX motif. Based on these data the putative protein encoded by pG-IF was tentatively called Drosophila lamin C. To address whether the pG-IF encoded protein is expressed and whether it encodes a cytoplasmic intermediate filament protein or a nuclear lamin we raised antibodies against the recombinant pG-IF protein. The antibodies decorate the nuclear envelope in Drosophila Kc tissue culture cells as well as in salivary and accessory glands demonstrating that pG-IF encodes a nuclear lamin (lamin C). Antibody decoration, in situ hybridization, western and northern blotting studies show that lamin C is acquired late in embryogenesis. In contrast, lamin Dm0 is constitutively expressed. Lamin C is first detected in late stage 12 embryos in oenocytes, hindgut and posterior spiracles and subsequently also in other differentiated tissues. In third instar larvae lamins C and Dm0 are coexpressed in all tissues tested. Thus, Drosophila has two lamins: lamin Dm0, containing a CaaX motif, is expressed throughout, while lamin C, lacking a CaaX motif, is expressed only later in development. Expression of Drosophila lamin C is similar to that of vertebrate lamin A (plus C), which loses its CaaX motif during incorporation into the lamina.

Interphase phosphorylation of the Drosophila nuclear lamin: site-mapping using a monoclonal antibody.

The Drosophila nuclear lamin is highly phosphorylated during interphase. Two interphase isoforms, differing in degree of phosphorylation, can be distinguished by one-dimensional SDS-polyacrylamide gel electrophoresis. One migrates with an apparent mass of 74 kDa (lamin Dm1); the other is more highly phosphorylated and migrates as a 76 kDa protein (lamin Dm2). We generated a monoclonal antibody, ADL84 which binds to lamin Dm1 but not lamin Dm2. Binding of ADL84 to lamin Dm2 was restored by phosphatase treatment of immunoblots containing lamins. Immunoprecipitation with ADL84 demonstrated that purified Drosophila nuclear lamins Dm1 and Dm2 are present as a random mixture of homo- and heterodimers. Indirect immunofluorescence experiments suggest that lamin Dm1 is present in all Drosophila cell types. The epitope for ADL84 was mapped by analyzing binding to bacterially expressed lamin deletion mutants and subsequently by screening for point mutants (randomly generated by polymerase chain reaction) which were not recognized by ADL84. The ADL84-epitope encompasses amino acids R22PPSAGP (arginine 22-proline 28). Peptide competition experiments demonstrated directly that phosphorylation of serine 25 impedes lamin binding by ADL84. This suggests that serine 25 is the lamin Dm2-specific phosphorylation site.