Sergei A. Golyshev’s research while affiliated with Lomonosov Moscow State University and other places

Electron microscopy (EM) is one of the most efficient methods for studying the fine structure of cells with a resolution thousands of times higher than that of visible light microscopy. The most advanced implementation of electron microscopy in biology is EM tomography of samples stabilized by freezing without water crystallization (cryoET). By circumventing the drawbacks of chemical fixation and dehydration, this technique allows investigating cellular structures in three dimensions at the molecular level, down to resolving individual proteins and their subdomains. However, the problem of efficient identification and localization of objects of interest has not yet been solved, thus limiting the range of targets to easily recognizable or abundant subcellular components. Labeling techniques provide the only way for locating the subject of investigation in microscopic images. CryoET imposes conflicting demands on the labeling system, including the need to introduce into a living cell the particles composed of substances foreign to the cellular chemistry that have to bind to the molecule of interest without disrupting its vital functions and physiology of the cell. This review examines both established and prospective methods for selective labeling of proteins and subcellular structures aimed to enable their localization in cryoET images.

Phytaspases, plant cell death-promoting and proprotein-processing proteolytic enzymes of the plant subtilase family, display aspartate (caspase-like) cleavage specificity and a very unusual retrograde trafficking from the apoplast to the cell interior upon induction of death-inducing stresses. To determine the underlying molecular mechanisms, we performed a search for tobacco phytaspase (NtPhyt) interactors using an in vivo cross-linking approach in Nicotiana tabacum plants. Tobacco Tubby-like F-box protein 8 (named Tubic hereafter) was identified as an NtPhyt interactor, with formation of the cross-linked complex being only efficient under the oxidative stress conditions. Direct interaction of the two proteins was further corroborated in the in vitro experiments. Analysis of Tubic-EGFP behavior in plant cells revealed that Tubic is a membrane-associated and fairly unstable protein. Furthermore, we showed that NtPhyt and Tubic are capable of negatively affecting one another in plant cells. On the other hand, down-regulation of Tubic in Tubic-silenced plants impaired specifically the retrograde transport of NtPhyt upon the induction of oxidative stress, testifying to a critical role of Tubic in this process. Our study, thus, contributes to understanding of the mechanisms of NtPhyt retrograde trafficking in plant cells subjected to stress.

Movement proteins (MPs) encoded by plant viruses are essential for cell-to-cell transport of viral genomes through plasmodesmata. The genome of hibiscus green spot virus contains a module of two MP genes termed ‘binary movement block’ (BMB), encoding the proteins BMB1 and BMB2. Here, BMB1 is shown to induce a defense response in Nicotiana benthamiana plants that inhibits BMB-dependent virus transport. This response is characterized by the accumulation of reactive oxygen species, callose deposition in the cell wall, and upregulation of 9-LOX expression. However, the BMB1-induced response is inhibited by coexpression with BMB2. Furthermore, BMB1 is found to localize to subnuclear structures, in particular to Cajal bodies, in addition to the cytoplasm. As shown in experiments with a BMB1 mutant, the localization of BMB1 to nuclear substructures enhances BMB-dependent virus transport. Thus, the virus transport mediated by BMB proteins is modulated by (i) a BMB1-induced defense response that inhibits transport, (ii) suppression of the BMB1-induced response by BMB2, and (iii) the nuclear localization of BMB1 that promotes virus transport. Collectively, the data presented demonstrate multiple levels of interactions between viral pathogens and their plant hosts during virus cell-to-cell transport.

Microtubules are an indispensable component of all eukaryotic cells due to their role in mitotic spindle formation, yet their organization and number can vary greatly in the interphase. The last common ancestor of all eukaryotes already had microtubules and microtubule motor proteins moving along them. Sponges are traditionally regarded as the oldest animal phylum. Their body does not have a clear differentiation into tissues, but it contains several distinguishable cell types. The choanocytes stand out among them and are responsible for creating a flow of water with their flagella and increasing the filtering and feeding efficiency of the sponge. Choanocyte flagella contain microtubules, but thus far, observing a developed system of cytoplasmic microtubules in non-flagellated interphase sponge cells has been mostly unsuccessful. In this work, we combine transcriptomic analysis, immunofluorescence, and electron microscopy with time-lapse recording to demonstrate that microtubules appear in the cytoplasm of sponge cells only when transdifferentiation processes are activated. We conclude that dynamic cytoplasmic microtubules in the cells of sponges are not a persistent but rather a transient structure, associated with cellular plasticity.

The recent advances achieved in microscopy technology have led to a significant breakthrough in biological research. Super-resolution fluorescent microscopy now allows us to visualize subcellular structures down to the pin-pointing of the single molecules in them, while modern electron microscopy has opened new possibilities in the study of protein complexes in their native, intracellular environment at near-atomic resolution. Nonetheless, both fluorescent and electron microscopy have remained beset by their principal shortcomings: the reliance on labeling procedures and severe sample volume limitations, respectively. Soft X-ray microscopy is a candidate method that can compensate for the shortcomings of both technologies by making possible observation of the entirety of the cellular interior without chemical fixation and labeling with an isotropic resolution of 40–70 nm. This will thus bridge the resolution gap between light and electron microscopy (although this gap is being narrowed, it still exists) and resolve the issue of compatibility with the former, and possibly in the near future, the latter methods. This review aims to assess the current state of soft X-ray microscopy and its impact on our understanding of the subcellular organization. It also attempts to look into the future of X-ray microscopy, particularly as relates to its seamless integration into the cell biology toolkit.

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs.

Soluble chaperones residing in the endoplasmic reticulum (ER) play vitally important roles in folding and quality control of newly synthesized proteins that transiently pass through the ER en route to their final destinations. These soluble residents of the ER are themselves endowed with an ER retrieval signal that enables the cell to bring the escaped residents back from the Golgi. Here, by using purified proteins, we showed that Nicotiana tabacum phytaspase, a plant aspartate-specific protease, introduces two breaks at the C-terminus of the N. tabacum ER resident calreticulin-3. These cleavages resulted in removal of either a dipeptide or a hexapeptide from the C-terminus of calreticulin-3 encompassing part or all of the ER retrieval signal. Consistently, expression of the calreticulin-3 derivative mimicking the phytaspase cleavage product in Nicotiana benthamiana cells demonstrated loss of the ER accumulation of the protein. Notably, upon its escape from the ER, calreticulin-3 was further processed by an unknown protease(s) to generate the free N-terminal (N) domain of calreticulin-3, which was ultimately secreted into the apoplast. Our study thus identified a specific proteolytic enzyme capable of precise detachment of the ER retrieval signal from a plant ER resident protein, with implications for the further fate of the escaped resident.

Liquid-liquid phase separation (LLPS) and liquid-solid phase transition (LSPT) of amyloidogenic proteins are now being intensively studied as a potentially widespread mechanism of pathological amyloids formation. However, the possibility and importance of such a mechanism in living systems is still questionable. Here, we investigated the possibility of such LSPT for a series of yeast prion proteins-based constructs overproduced in yeast cells lacking any pre-existing amyloid template. By combining fluorescence and electron microscopy with biochemical and genetic approaches, we have shown that three such constructs (containing the prion domains (PDs) of either Sup35, Rnq1 or Mot3 proteins) form amyloid fibrils via the intermediate stage of liquid-like condensates, that age over time into the more solid-like hydrogels and amyloid bodies. In turn, LSPT of these constructs triggers prion conversion of the corresponding wild-type protein. Two other constructs studied (Ure2- and Sap30-based) are unable to phase separate in vivo and their amyloidogenesis is therefore strongly suppressed. Using PrK-resistant amyloid core mapping, we showed that Sup35PD amyloids formed via LSPT have a different molecular architecture compared to those formed via amyloid cross-seeding. Finally, we showed that physiological LLPS of wild-type Sup35 protein can increase its prion conversion in yeast.