Biomedical subjects

Federica Brandizzi

Publications and source records attributed to Federica Brandizzi.

At least 19 recordsLinked to original sources

PubMed2025

Through the lens of bioenergy crops: advances, bottlenecks, and promises of plant engineering.

Advances in engineering of bioenergy crops were driven over the past years by adapting technological breakthroughs and accelerating conventional applications but also exposed intriguing challenges. New tools revealed rich interconnectivity in the exponentially growing and dynamic 'big' omics data' of metabolomes, transcriptomes, and genomes at previously inaccessible magnitude (global, cross-species, meta-) and resolution (single cell). Insights enabled fresh hypotheses and stimulated disciplines such as functional genomics with discovery of broad regulatory networks and their determinants, that is, DNA parts, including promoters, regulatory elements, and transcription factors. Their rational design, assembly into increasingly complex blueprints, and installation into diverse chassis is an existing frontier that may benefit from emerging technologies to address bottlenecks. Interweaving nature-inspired to fully synthetic parts has already allowed building of fine-tuned regulatory circuits, or new-to-nature metabolic routes insulated from the biological context of the chassis species. Similarly, developments and the evolving need for unifying principles in plant transformation and species-agnostic technologies highlight future opportunities for engineering the next generation of bioenergy plants.

Crops, Agricultural↗

PubMed2006

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond.

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.

Endoplasmic Reticulum↗

PubMed2006

The plant ER-Golgi interface: a highly structured and dynamic membrane complex.

As compared with other eukaryotic cells, plants have developed an endoplasmic reticulum (ER)-Golgi interface with very specific structural characteristics. ER to Golgi and Golgi to ER transport appear not to be dependent on the cytoskeleton, and ER export sites have been found closely associated with Golgi bodies to constitute entire mobile units. However, the molecular machinery involved in membrane trafficking seems to be relatively conserved among eukaryotes. Therefore, a challenge for plant scientists is to determine how these molecular machineries work in a different structural and dynamic organization. This review will focus on some aspects of membrane dynamics that involve coat proteins, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptor proteins), lipids, and lipid-interacting proteins.

Endoplasmic Reticulum↗

PubMed2006

Molecular control of nuclear and subnuclear targeting of the plant CDK inhibitor ICK1 and ICK1-mediated nuclear transport of CDKA.

ICK1 is the first member of a family of plant cyclin-dependent kinase (CDK) inhibitors. It has been shown that ICK1 is localized in the nuclei of transgenic Arabidopsis plants. Since cellular localization is important for the functions of cell cycle regulators, a comprehensive analysis was undertaken to identify specific sequences regulating the cellular localization of ICK1. Deletion and site-specific mutants fused to the green fluorescent protein (GFP) were used in transgenic Arabidopsis plants and transfected tobacco cells. Surprisingly, three separate sequences in the N-terminal, central and C-terminal regions of ICK1 could independently confer nuclear localization of the GFP fusion proteins. The central nuclear localization signal NLS(ICK1) could transport the much larger GUS (beta-glucuronidase)-GFP fusion protein into nuclei, while the other two sequences were unable to. These results suggest that NLS(ICK1) is a strong NLS that actively transports the fusion protein into nuclei, while the other two sequences are either a weaker NLS or confer the nuclear localization of GFP indirectly. It was further observed that the N-terminal sequence specifies a punctate pattern of subnuclear localization, while the C-terminal sequence suppresses it. Furthermore, co-expression of ICK1 and Arabidopsis CDKA, tagged with different GFP variants, showed that ICK1 could mediate the transport of CDKA into nuclei while a mutant ICK1(1-162) that does not interact with CDKA lost this ability. These results illustrate how the nuclear localization of ICK1 is regulated and also suggest a possible role of ICK1 in regulating the cellular distribution of CDKA.

Arabidopsis↗

PubMed2006

Seeking a way out: export of proteins from the plant endoplasmic reticulum.

The functionality of the secretory pathway relies on the efficient transfer of cargo molecules from their site of synthesis in the endoplasmic reticulum (ER) to successive compartments within the pathway. Although transport mechanisms of secretory proteins have been studied in detail in various non-plant systems, it is only recently that our knowledge of secretory routes in plants has expanded dramatically. This review focuses on exciting new findings concerning the exit mechanisms of cargo proteins from the plant ER and the role of ER export sites in this process.

Animals↗

PubMed2006

Mapping the Arabidopsis organelle proteome.

A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.

Arabidopsis↗

PubMed2006

Fluorescent proteins as markers in the plant secretory pathway.

The use of fluorescent proteins and live cell imaging has greatly increased our knowledge of cell biology in recent years. Not only can these technologies be used to study protein trafficking under different conditions, but they have also been of use in elucidating the relationships between different organelles in a noninvasive manner. The use of multiple different fluorochromes allows the observation of interactions between organelles and between proteins, making this one of the fastest-developing and exciting fields at this time. In this review, we discuss the multitude of fluorescent markers that have been generated to study the plant secretory pathway. Although these markers have been used to solve many mysteries in this field, some areas that require further discussion remain.

Arabidopsis Proteins↗

PubMed2006

ARL1 plays a role in the binding of the GRIP domain of a peripheral matrix protein to the Golgi apparatus in plant cells.

ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.

Amino Acid Motifs↗

PubMed2006

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

ADP-Ribosylation Factor 1↗

PubMed2005

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells.

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.

Biomarkers↗

PubMed2005

Diacidic motifs influence the export of transmembrane proteins from the endoplasmic reticulum in plant cells.

In yeast and mammals, amino acid motifs in the cytosolic tails of transmembrane domains play a role in protein trafficking by facilitating export from the endoplasmic reticulum (ER). However, little is known about ER export signals of membrane proteins in plants. Therefore, we investigated the role of diacidic motifs in the ER export of Golgi-localized membrane proteins. We show that diacidic motifs perform a significant function in the export of transmembrane proteins to the Golgi apparatus, as mutations of these signals impede the efficient anterograde transport of multispanning, type II, and type I proteins. Furthermore, we demonstrate that diacidic motifs instigate the export of proteins that reside in the ER due to the lengths of their transmembrane domains. However, not all of the diacidic motifs in the cytosolic tails of the proteins studied were equally important in ER export. Transport of Golgi proteins was disrupted only by mutagenesis of specific diacidic signals, suggesting that the protein environment of these signals affects their function. Our findings indicate that diacidic ER export motifs are present and functional in plant membrane proteins and that they are dominant over transmembrane domain length in determining the export of proteins from the ER in plant cells.

Amino Acid Motifs↗

PubMed2005

Constitutive nuclear localization of activated caspase 3 in subpopulations of the astroglial family of cells.

In a study examining for apoptosis in spontaneously hypertensive rats (SHR), we observed the constitutive presence of activated caspase 3 in Bergmann glia. We then examined stroke-prone SHR as well as the normotensive strains Wistar, Wistar Kyoto, and Sprague-Dawley. In all these strains, we found that Bergmann glia expressed activated caspase 3 in nuclei. Furthermore, subpopulations of astrocytes in the granular layer of the cerebellar cortex, in the hippocampus, and spinal cord gray matter, particularly in the dorsal part of the dorsal horns, expressed nuclear activated caspase 3. This distribution corresponds to the distribution of astrocytes that express the glutamate transporter GLAST. We conclude that Bergmann glia and a subpopulation of astrocytes throughout the CNS express activated caspase 3 in nuclei to fulfill a yet-to-be defined non-apoptotic function. GLIA.

Amino Acid Transport System X-AG↗

PubMed2005

Identification and characterization of AtCASP, a plant transmembrane Golgi matrix protein.

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.

Amino Acid Motifs↗

PubMed2005

Receptor salvage from the prevacuolar compartment is essential for efficient vacuolar protein targeting.

We have characterized the requirements to inhibit the function of the plant vacuolar sorting receptor BP80 in vivo and gained insight into the crucial role of receptor recycling between the prevacuolar compartment and the Golgi apparatus. The drug wortmannin interferes with the BP80-mediated route to the vacuole and induces hypersecretion of a soluble BP80-ligand. Wortmannin does not prevent receptor-ligand binding itself but causes BP80 levels to be limiting. Consequently, overexpression of BP80 partially restores vacuolar cargo transport. To simulate receptor traffic, we tested a truncated BP80 derivative in which the entire lumenal domain of BP80 has been replaced by the green fluorescent protein (GFP). The resulting chimeric protein (GFP-BP80) accumulates in the prevacuolar compartment as expected, but a soluble GFP fragment can also be detected in purified vacuoles. Interestingly, GFP-BP80 coexpression interferes with the correct sorting of a BP80-ligand and causes hypersecretion that is reversible by expressing a 10-fold excess of full-length BP80. This suggests that GFP-BP80 competes with endogenous BP80 mainly at the retrograde transport route that rescues receptors from the prevacuolar compartment. Treatment with wortmannin causes further leakage of GFP-BP80 from the prevacuolar compartment to the vacuoles, whereas BP80-ligands are secreted. We propose that recycling of the vacuolar sorting receptor from the prevacuolar compartment to the Golgi apparatus is an essential process that is saturable and wortmannin sensitive.

Carrier Proteins↗

PubMed2005

Improved transcriptional activators and their use in mis-expression traps in Arabidopsis.

The synthetic transcription factor LhG4 has been used in numerous mis-expression studies in plants. We show that the sequence encoding the LhG4 activation domain, derived from Saccharomyces cerevisiae GAL4, contains several cryptic polyadenylation signals in Arabidopsis. The GAL4-derived sequence was modified according to preferred Arabidopsis codon usage, generating LhG4AtO which was faithfully transcribed in Arabidopsis plants. In protoplasts, LhG4AtO achieved maximum transactivation of the pOp promoter with 10-fold less input DNA than LhG4. The same methods were used to compare 10 other LhG4 derivatives that carried alternative natural or synthetic activation domains. Lh214 and Lh314, which contain synthetic activation domains comprising trimers of a core acidic activation domain, directed threefold more GUS expression from the pOp promoter with 20-fold less input DNA than LhG4. In contrast, when expressed from the CaMV 35S promoter in transgenic plants carrying a pOp-GUS reporter, Lh214 and Lh314 yielded transformants with substantially lower GUS activities than other constructs including LhG4AtO and LhG4 which performed similarly. When incorporated into an enhancer-trapping vector, however, LhG4AtO and Lh314 yielded enhancer traps with approximately twice the frequency of LhG4, suggesting that the modified activation domains offer improved performance when expressed from weaker transcription signals. To increase the number of LhG4 patterns available for mis-expression studies, we describe a population of enhancer-trap lines obtained with LhG4AtO in a pOp-GUS background. We show that enhancer-trap lines can transactivate an unlinked pOp-green fluorescent protein (pOp-GFP) reporter in the pattern predicted by staining for GUS activity.

Amino Acid Sequence↗

PubMed2005

Crossing the divide--transport between the endoplasmic reticulum and Golgi apparatus in plants.

The transport of proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in plants is an exciting and constantly expanding topic, which has attracted much attention in recent years. The study of protein transport within the secretory pathway is a relatively new field, dating back to the 1970s for mammalian cells and considerably later for plants. This may explain why COPI- and COPII-mediated transport between the ER and the Golgi in plants is only now becoming clear, while the existence of these pathways in other organisms is relatively well documented. We summarize current knowledge of these protein transport routes, as well as highlighting key differences between those of plant systems and those of mammals and yeast. These differences have necessitated the study of plant-specific aspects of protein transport in the early secretory pathway, and this review discusses recent developments in this area. Advances in live-cell-imaging technology have allowed the observation of protein movement in vivo, giving a new insight into many of the processes involved in vesicle formation and protein trafficking. The use of these new technologies has been combined with more traditional methods, such as protein biochemistry and electron microscopy, to increase our understanding of the transport routes in the cell.

Animals↗

PubMed2004

AtRabF2b (Ara7) acts on the vacuolar trafficking pathway in tobacco leaf epidermal cells.

Rab GTPases are universal key regulators of intracellular secretory trafficking events. In particular, Rab 5 homologues have been implicated in endocytic events and in the vacuolar pathway. In this study, we investigate the location and function of a member of this family, AtRabF2b (Ara7) in tobacco (Nicotiana tabacum) leaf epidermal cells using a live cell imaging approach. Fluorescent-tagged AtRabF2b[wt] localized to the prevacuolar compartment and Golgi apparatus, as determined by coexpression studies with fluorescent markers for these compartments. Mutations that impair AtRabF2b function also alter the subcellular location of the GTPase. In addition, coexpression studies of the protein with the vacuole-targeted aleurain-green fluorescent protein (GFP) and rescue experiments with wild-type AtRabF2b indicate that the dominant-negative mutant of AtRabF2b causes the vacuolar marker to be secreted to the apoplast. Our results indicate a clear role of AtRabF2b in the vacuolar trafficking pathway.

Biomarkers↗

PubMed2004

Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells.

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.

Agrobacterium tumefaciens↗