Multiple C2 domains and transmembrane region proteins (MCTPs) tether membranes at plasmodesmata.

In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.

Plasma Membrane-Associated Receptor-like Kinases Relocalize to Plasmodesmata in Response to Osmotic Stress.

Plasmodesmata act as key elements in intercellular communication, coordinating processes related to plant growth, development, and responses to environmental stresses. While many of the developmental, biotic, and abiotic signals are primarily perceived at the plasma membrane (PM) by receptor proteins, plasmodesmata also cluster receptor-like activities; whether these two pathways interact is currently unknown. Here, we show that specific PM-located Leu-rich-repeat receptor-like-kinases, Qian Shou kinase (QSK1) and inflorescence meristem kinase2, which under optimal growth conditions are absent from plasmodesmata, rapidly relocate and cluster to the pores in response to osmotic stress. This process is remarkably fast, is not a general feature of PM-associated proteins, and is independent of sterol and sphingolipid membrane composition. Focusing on QSK1, previously reported to be involved in stress responses, we show that relocalization in response to mannitol depends on QSK1 phosphorylation. Loss-of-function mutation in QSK1 results in delayed lateral root (LR) development, and the mutant is affected in the root response to mannitol stress. Callose-mediated plasmodesmata regulation is known to regulate LR development. We found that callose levels are reduced in the qsk1 mutant background with a root phenotype resembling ectopic expression of PdBG1, an enzyme that degrades callose at the pores. Both the LR and callose phenotypes can be complemented by expression of wild-type and phosphomimic QSK1 variants, but not by phosphodead QSK1 mutant, which fails to relocalize at plasmodesmata. Together, the data indicate that reorganization of receptor-like-kinases to plasmodesmata is important for the regulation of callose and LR development as part of the plant response to osmotic stress.

Organic matrices in metazoan calcium carbonate skeletons: Composition, functions, evolution.

Calcium carbonate skeletal tissues in metazoans comprise a small quantity of occluded organic macromolecules, mostly proteins and polysaccharides that constitute the skeletal matrix. Because its functions in modulating the biomineralization process are well known, the skeletal matrix has been extensively studied, successively via classical biochemical approaches, via molecular biology and, in recent years, via transcriptomics and proteomics. The optimistic view that the deposition of calcium carbonate minerals requires a limited number of macromolecules has been challenged, in the last decade, by high-throughput approaches. Such approaches have made possible the rapid identification of large sets of mineral-associated proteins, i.e., 'skeletal repertoires' or 'skeletomes', in several calcifying animal models, ranging from sponges to echinoderms. One of the consequences of this expanding set of data is that a simple definition of the skeletal matrix is no longer possible. This increase in available data, however, makes it easier to compare skeletal repertoires, shedding light on the fundamental evolutionary mechanisms affecting matrix components.

Insight into the primary mode of action of TiO2 nanoparticles on Escherichia coli in the dark.

Large-scale production and incorporation of titanium dioxide nanoparticles (NP-TiO2 ) in consumer products leads to their potential release into the environment and raises the question of their toxicity. The bactericidal mechanism of NP-TiO2 under UV light is known to involve oxidative stress due to the generation of reactive oxygen species. In the dark, several studies revealed that NP-TiO2 can exert toxicological effects. However, the mode of action of these nanoparticles is still controversial. In the present study, we used a combination of fluorescent probes to show that NP-TiO2 causes Escherichia coli membrane depolarization and loss of integrity, leading to higher cell permeability. Using both transcriptomic and proteomic global approaches we showed that this phenomenon translates into a cellular response to osmotic stress, metabolism of cell envelope components and uptake/metabolism of endogenous and exogenous compounds. This primary mechanism of bacterial NP-TiO2 toxicity is supported by the observed massive cell leakage of K(+) /Mg(2+) concomitant with the entrance of extracellular Na(+), and by the depletion of intracellular ATP level.

Shell proteome of rhynchonelliform brachiopods.

Brachiopods are a phylum of marine invertebrates that have an external bivalved shell to protect their living tissues. With few exceptions, this biomineralized structure is composed of calcite, mixed together with a minor organic fraction, comprising secreted proteins that become occluded in the shell structure, once formed. This organic matrix is thought to display several functions, in particular, to control mineral deposition and to regulate crystallite shapes. Thus, identifying the primary structure of matrix proteins is a prerequisite for generating bioinspired materials with tailored properties. In this study, we employed a proteomic approach to identify numerous peptides that constitute the shell proteins, in three rhynchonellid brachiopods from different localities. Our results suggest that the shell protein repertoires identified thus far, differ from that of better known calcifying metazoans, such as molluscs.

Molecular characterization of accripin11, a soluble shell protein with an acidic C-terminus, identified in the prismatic layer of the Mediterranean fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

A yeast toxic mutant of HET-s((218-289)) prion displays alternative intermediates of amyloidogenesis.

Amyloids are thought to be involved in various types of neurodegenerative disorders. Several kinds of intermediates, differing in morphology, size, and toxicity, have been identified in the multistep amyloidogenesis process. However, the mechanisms explaining amyloid toxicity remain unclear. We previously generated a toxic mutant of the nontoxic HET-s((218-289)) amyloid in yeast. Here we report that toxic and nontoxic amyloids differ not only in their structures but also in their assembling process. We used multiple and complementary methods to investigate the intermediates formed by these two amyloids. With the methods used, no intermediates were observed for the nontoxic amyloid; however, under the same experimental conditions, the toxic mutant displayed visible oligomeric and fibrillar intermediates.

Driving amyloid toxicity in a yeast model by structural changes: a molecular approach.

The amyloid aggregation pathway is a multistep process, and many in vitro studies have highlighted the role of particular intermediates in the cellular toxicity of various amyloid diseases. In a previous study, we generated a yeast toxic mutant (M8) of the harmless model amyloid protein Het-s(218-289). In this study, we compared the aggregation characteristics of the wild-type (WT) and the toxic mutant at the molecular level. Both proteins formed fibrillar amyloid aggregates but with different dye-binding properties and X-ray diffraction patterns. The toxic amyloid formed very unusual short (80 nm) unbranched fibers visible on transmission electron microscopy. Fourier transform infrared spectroscopy demonstrated that M8 beta-sheets were essentially organized into a mixed parallel and antiparallel structure, whereas the WT protein displayed a predominantly parallel organization. Cellular toxicity may therefore be related to assembly of the toxic amyloid in a new aggregation pathway.

The polyphenol piceid destabilizes preformed amyloid fibrils and oligomers in vitro: hypothesis on possible molecular mechanisms.

Alzheimer's disease (AD) is characterized by deposits of amyloid in various tissues. The neuronal cytotoxicity of Abeta peptides is attributed not only to various mechanisms but also to amyloid fibrils and soluble oligomeric intermediates. Consequently, finding molecules to prevent or reverse the oligomerization and fibrillization of Abeta could be of therapeutic value in the treatment of AD. We show that piceid, a polyphenol of the stilbene family, destabilized fibrils and oligomers to give back monomers that are not neurotoxic molecules. The mechanism of this destabilization could be a dynamic interaction between the polyphenol and the Abeta that could open the hydrophobic zipper and shift the reversible equilibrium "random coil<-->beta-sheet" to the disordered structure.

Lipids or Proteins: Who Is Leading the Dance at Membrane Contact Sites?

Understanding the mode of action of membrane contact sites (MCSs) across eukaryotic organisms at the near-atomic level to infer function at the cellular and tissue levels is a challenge scientists are currently facing. These peculiar systems dedicated to inter-organellar communication are perfect examples of cellular processes where the interplay between lipids and proteins is critical. In this mini review, we underline the link between membrane lipid environment, the recruitment of proteins at specialized membrane domains and the function of MCSs. More precisely, we want to give insights on the crucial role of lipids in defining the specificity of plant endoplasmic reticulum (ER)-plasma membrane (PM) MCSs and we further propose approaches to study them at multiple scales. Our goal is not so much to go into detailed description of MCSs, as there are numerous focused reviews on the subject, but rather try to pinpoint the critical elements defining those structures and give an original point of view by considering the subject from a near-atomic angle with a focus on lipids. We review current knowledge as to how lipids can define MCS territories, play a role in the recruitment and function of the MCS-associated proteins and in turn, how the lipid environment can be modified by proteins.

Proteomic Analysis of Lipid Droplets from Arabidopsis Aging Leaves Brings New Insight into Their Biogenesis and Functions.

Lipid droplets (LDs) are cell compartments specialized for oil storage. Although their role and biogenesis are relatively well documented in seeds, little is known about their composition, structure and function in senescing leaves where they also accumulate. Here, we used a label free quantitative mass spectrometry approach to define the LD proteome of aging Arabidopsis leaves. We found that its composition is highly different from that of seed/cotyledon and identified 28 proteins including 9 enzymes of the secondary metabolism pathways involved in plant defense response. With the exception of the TRIGALACTOSYLDIACYLGLYCEROL2 protein, we did not identify enzymes implicated in lipid metabolism, suggesting that growth of leaf LDs does not occur by local lipid synthesis but rather through contact sites with the endoplasmic reticulum (ER) or other membranes. The two most abundant proteins of the leaf LDs are the CALEOSIN3 and the SMALL RUBBER PARTICLE1 (AtSRP1); both proteins have structural functions and participate in plant response to stress. CALEOSIN3 and AtSRP1 are part of larger protein families, yet no other members were enriched in the LD proteome suggesting a specific role of both proteins in aging leaves. We thus examined the function of AtSRP1 at this developmental stage and found that AtSRP1 modulates the expression of CALEOSIN3 in aging leaves. Furthermore, AtSRP1 overexpression induces the accumulation of triacylglycerol with an unusual composition compared to wild-type. We demonstrate that, although AtSRP1 expression is naturally increased in wild type senescing leaves, its overexpression in senescent transgenic lines induces an over-accumulation of LDs organized in clusters at restricted sites of the ER. Conversely, atsrp1 knock-down mutants displayed fewer but larger LDs. Together our results reveal that the abundancy of AtSRP1 regulates the neo-formation of LDs during senescence. Using electron tomography, we further provide evidence that LDs in leaves share tenuous physical continuity as well as numerous contact sites with the ER membrane. Thus, our data suggest that leaf LDs are functionally distinct from seed LDs and that their biogenesis is strictly controlled by AtSRP1 at restricted sites of the ER.

Investigating the emerging role of comparative proteomics in the search for new biomarkers of metal contamination under varying abiotic conditions.

This study aims at investigating the potential use of comparative proteomics as a multi-marker approach of metal contamination, taking into account the potential confounding effect of water temperature. The major objective was to identify combinations of proteins specifically responding to a given metal, even if included in a metal mixture. The diagnostic approach was performed via the comparative analysis of protein expression on spot mapping provided by adult males of Gammarus pulex (Amphipoda, Crustacea) respectively exposed to arsenate (As), cadmium (Cd) or a binary mixture of these metals (AsCd) at three realistic temperatures (5, 10 and 15°C). Proteomic expression analysis was performed by Differential in-Gel Electrophoresis (2D-DiGE), and completed by an adapted inferential statistical approach. Combinations of under/over-expressed protein spots discriminated the metal identity. However, none of these spots discriminated both the individual metal effect (As or Cd) and its effect in metal mixture (AsCd) whatever the tested temperature. Some limits of the two-dimensional analysis of protein spot maps in G. pulex have been highlighted: (i) the presence of contaminating peptides and/or abundant "déja-vu" proteins which can mask the responses of other proteins of interest or (ii) the presence of post-translational modifications. An optimization of the experimental design (especially during the sample preparation) has been described for future investigations. This study has also highlighted (i) the importance of precisely identifying the protein spots of interest to avoid erroneous interpretations in terms of action mechanisms of chemicals and (ii) the importance of working under controlled laboratory conditions with a temperature close to 10°C. In such conditions, we have demonstrated a higher impact of As than Cd on the energetic metabolism of Gammarus. This As impact is reduced in AsCd mixture confirming the antagonistic interaction of this binary mixture previously observed on G. pulex mortality at 10°C.

The Shell of the Invasive Bivalve Species Dreissena polymorpha: Biochemical, Elemental and Textural Investigations.

The zebra mussel Dreissena polymorpha is a well-established invasive model organism. Although extensively used in environmental sciences, virtually nothing is known of the molecular process of its shell calcification. By describing the microstructure, geochemistry and biochemistry/proteomics of the shell, the present study aims at promoting this species as a model organism in biomineralization studies, in order to establish a bridge with ecotoxicology, while sketching evolutionary conclusions. The shell of D. polymorpha exhibits the classical crossed-lamellar/complex crossed lamellar combination found in several heterodont bivalves, in addition to an external thin layer, the characteristics of which differ from what was described in earlier publication. We show that the shell selectively concentrates some heavy metals, in particular uranium, which predisposes D. polymorpha to local bioremediation of this pollutant. We establish the biochemical signature of the shell matrix, demonstrating that it interacts with the in vitro precipitation of calcium carbonate and inhibits calcium carbonate crystal formation, but these two properties are not strongly expressed. This matrix, although overall weakly glycosylated, contains a set of putatively calcium-binding proteins and a set of acidic sulphated proteins. 2D-gels reveal more than fifty proteins, twenty of which we identify by MS-MS analysis. We tentatively link the shell protein profile of D. polymorpha and the peculiar recent evolution of this invasive species of Ponto-Caspian origin, which has spread all across Europe in the last three centuries.

A minimal molecular toolkit for mineral deposition? Biochemistry and proteomics of the test matrix of adult specimens of the sea urchin Paracentrotus lividus.

UNLABELLED: The sea urchin endoskeleton consists of a magnesium-rich biocalcite comprising a small amount of occluded organic macromolecules. This structure constitutes a key-model for understanding the mineral--organics interplay, and for conceiving in vitro bio-inspired materials with tailored properties. Here we employed a deep-clean technique to purify the occluded proteins from adult Paracentrotus lividus tests. We characterized them by 1- and 2D-electrophoreses, ELISA and immunoblotting, and using liquid chromatography coupled with Mass Spectrometry (nanoLC-MS/MS), we identified two metalloenzymes (carbonic anhydrase and MMP), a set of MSP130 family members, several C-type lectins (SM29, SM41, PM27) and cytoskeletal proteins. We demonstrate the effect of the protein extract on the crystals, with an in vitro crystallization assay. We suggest that this small set of biomineralization proteins may represent a 'minimal molecular crystallization toolkit'. SIGNIFICANCE: Biominerals often exhibit superior chemical properties, when compared to their inorganic counterparts. This is due pro parte to the proteins that are occluded in the mineral. However, the limited available studies on biomineralization have not yet succeeded in identifying a minimal set of proteins directly involved in the formation of the biomineral in vivo and sufficiently required for in vitro precipitation. Indeed, the high number of proteins identified by high-throughput screening in the recent years does not encourage the possibility of recreating or tailoring the mineral in vitro. Thus, the identification of biomineralization proteins involved in protein-mineral interactions is highly awaited. In the present study, we used the sea urchin, Paracentrotus lividus (P. lividus), to identify the native proteins directly taking part in protein-mineral interactions. We employed an improved deep-clean technique to extract and purify the native occluded skeletal matrix proteins from the test and identified them by the highly sensitive technique of nanoLC-MS/MS. We show that this minimal set of proteins has a shaping effect on the formation of biocalcite in vitro. This work gives insights on the biomineralization of the sea urchin, while it paves the way for the identification of biomineralization proteins in other biomineralizing systems. Understanding the 'biologically controlled mineralization' will facilitate the in vitro formation and tailoring of biominerals in mild conditions for applications in medicine and materials science.

Spine and test skeletal matrices of the Mediterranean sea urchin Arbacia lixula--a comparative characterization of their sugar signature.

Calcified structures of sea urchins are biocomposite materials that comprise a minor fraction of organic macromolecules, such as proteins, glycoproteins and polysaccharides. These macromolecules are thought to collectively regulate mineral deposition during the process of calcification. When occluded, they modify the properties of the mineral. In the present study, the organic matrices (both soluble and insoluble in acetic acid) of spines and tests from the Mediterranean black sea urchin Arbacia lixula were extracted and characterized, in order to determine whether they exhibit similar biochemical signatures. Bulk characterizations were performed by mono-dimensional SDS/PAGE, FT-IR spectroscopy, and an in vitro crystallization assay. We concentrated our efforts on characterization of the sugar moieties. To this end, we determined the monosaccharide content of the soluble and insoluble organic matrices of A. lixula spines and tests by HPAE-PAD, together with their respective lectin-binding profiles via enzyme-linked lectin assay. Finally, we performed in situ localization of N-acetyl glucosamine-containing saccharides on spines and tests using gold-conjugated wheatgerm agglutinin. Our data show that the test and spine matrices exhibit different biochemical signatures with regard to their saccharidic fraction, suggesting that future studies should analyse the regulation of mineral deposition by the matrix in these two mineralized structures in detail. This study re-emphasizes the importance of non-protein moieties, i.e. sugars, in calcium carbonate systems, and highlights the need to clearly identify their function in the biomineralization process.

The test skeletal matrix of the black sea urchin Arbacia lixula.

In the field of biomineralization, the past decade has been marked by the increasing use of high throughput techniques, i.e. proteomics, for identifying in one shot the protein content of complex macromolecular mixtures extracted from mineralized tissues. Although crowned with success, this approach has been restricted so far to a limited set of key-organisms, such as the purple sea urchin Strongylocentrotus purpuratus, the pearl oyster or the abalone, leaving in the shadow non-model organisms. As a consequence, it is still unknown to what extent the calcifying repertoire varies, from group to group, at high (phylum, class), median (order, family) or low (genus, species) taxonomic rank. The present paper shows the first biochemical and proteomic characterization of the test matrix of the Mediterranean black sea urchin Arbacia lixula (Arbacioida). Our work suggests that the skeletal repertoire of A. lixula exhibits some similarities but also several differences with that of the few sea urchin species (S. purpuratus, Paracentrotus lividus), for which molecular data are already available. The differences may be attributable to the taxonomic position of the species considered: A. lixula belongs to an order - Arbacioida - that diverged more than one hundred million years ago from the Camarodonta, which includes the two species S. purpuratus and P. lividus. For the echinoid class, we suggest that large-scale proteomic screening should be performed in order to understand which molecular functions related to calcification are conserved and which ones have been co-opted for biomineralization in particular lineages.

Effect of deltamethrin (pyrethroid insecticide) on two clones of Daphnia magna (Crustacea, Cladocera): a proteomic investigation.

Deltamethrin is a class II pyrethroid insecticide commonly used in agriculture. It is hazardous to freshwater ecosystems, especially for the cladoceran Daphnia magna (Straus 1820). The results of our previous studies based on acute and chronic ecotoxicity experiments revealed differences in the sensitivity between two different clones. In this work, to investigate deltamethrin toxicity mechanisms in two clones of D. magna, we used a proteomic approach in order to analyze changes in protein expression profiles after 48 h of exposure. We detected 1339 spots; then applying statistical criteria (ANOVA p<0.001 and minimum fold change 1.5), only 128 spots were significantly different in the normalized volume. Among the preselected proteins there were 88 up-regulated and 40 down-regulated proteins. Results showed differences in sensitivities after deltamethrin exposure between the clones. Moreover, using the 2-DIGE method, proteomic investigation for deltamethrin exposure proved to be a reliable and powerful approach to investigate effects of deltamethrin as part of research for new metabolic and cellular biomarkers. After identification by mass spectrometry, there were 39 proteins recognized and identified, in which 21 and 18 were up- and down-regulated, respectively, in deltamethrin-exposed clone A compared to three other conditions (controls of each clone and deltamethrin-exposed clone 2). Up- and down-regulated proteins belonged to 12 biological processes (i.e. metabolic processes, apoptosis and stimulus response) and 5 molecular functions (i.e. catalytic activity, binding, structural molecular activity, antioxidant and receptor activities). Identification of these deregulated proteins opens a new way in discovering new molecular targets and putative biomarkers in daphnids exposed to deltamethrin.

Physiological response and differential leaf proteome pattern in the European invasive Asteraceae Solidago canadensis colonizing a former cokery soil.

Derelict contaminated sites are often colonized spontaneously by plant species leading to a vegetal cover thought to limit particle dispersal and polluted water infiltration. Those plants must cope with soil pollutants through tolerance mechanisms that are not yet fully understood. Here, we focused our attention on a particular Asteraceae plant, Solidago canadensis, considered as invasive in Europe. S. canadensis spontaneously growing on either polluted (NM soil) or control soils dumped on experimental plots were studied for their physiological status, oxidative stress and 2D-DIGE of leaf extracts. S. canadensis tolerance to soil pollutants was demonstrated since growth rates, allocation to reproduction ratios and Fv/Fm ratios were similar in plants from control and NM soil. At the cell level, the catalase activity level was increased in plants collected on NM soil while lipoperoxidation was unaffected. Also, the leaf proteomic study revealed thirty down-regulated and sixty-six up-regulated proteins. Abundances of proteins related to oxidative stress, carbohydrate metabolism, ion transport were mainly up-regulated while those of proteins involved in cell cycle and transcription/translation were mostly down-regulated. Proteins associated to protein metabolism were either down- or up-regulated. Considered altogether, we highlighted that S. canadensis exhibited a complex proteome response when experiencing a multicontaminated soil.

Comparison of arsenate and cadmium toxicity in a freshwater amphipod (Gammarus pulex).

Cadmium is largely documented on freshwater organisms while arsenic, especially arsenate, is rarely studied. The kinetic of the LC50s values for both metals was realized on Gammarus pulex. Physiological [i.e. metal concentration in body tissues, bioconcentration factor (BCF)] effects and behavioural responses (via pleopods beats) were investigated after 240-h exposure. Arsenate LC50 value was 100 fold higher than Cd-LC50 value after 240-h exposure, while concentrations in gammarids were similar for both metals at their respective LC50s. BCF decreased with increasing cadmium concentration while BCF remained stable with increasing arsenate concentration. Moreover, BCF was between 148 and 344 times lower for arsenate than cadmium. A significant hypoventilation was observed for cadmium concentrations exceeding or close to the 240h-LC50(Cd), while gammarids hyperventilated for the lowest arsenate concentrations and hypoventilated for the highest arsenate concentrations. We discussed the relationships between potential action mechanisms of these two metals and observed results.

Screening for toxic amyloid in yeast exemplifies the role of alternative pathway responsible for cytotoxicity.

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species.