Protocol for automated multivariate quantitative-image-based cytometry analysis by fluorescence microscopy of asynchronous adherent cells.

Here, we present a protocol for multivariate quantitative-image-based cytometry (QIBC) analysis by fluorescence microscopy of asynchronous adherent cells. We describe steps for the preparation, treatment, and fixation of cells, sample staining, and imaging for QIBC. We then detail image analysis with our open source Fiji script developed for QIBC and present multiparametric data visualization. Our QIBC Fiji script integrates modern artificial-intelligence-based tools, applying deep learning, for robust automated nuclei segmentation with minimal user adjustments, a major asset for efficient QIBC analysis. For complete details on the use and execution of this protocol, please refer to Besse et al. (2023).1.

A pulse-chase strategy combining click-EdU and photoconvertible fluorescent reporter: tracking Golgi protein dynamics during the cell cycle.

Imaging or quantifying protein synthesis in cellulo through a well-resolved analysis of the cell cycle (also defining G1 subcompartments) is a methodological challenge. Click chemistry is the method of choice to reveal the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) and track proliferating nuclei undergoing DNA synthesis. However, the click reaction quenches fluorescent proteins. Our challenge was to reconcile these two tools. A robust protocol based on a high-resolution cytometric cell cycle analysis in tobacco (Nicotiana tabacum) BY2 cells expressing fluorescent Golgi markers has been established. This was broadly applicable to tissues, cell clusters, and other eukaryotic material, and compatible with Scale clearing. EdU was then used with the photoconvertible protein sialyl transferase (ST)-Kaede as a Golgi marker in a photoconversion pulse-chase cytometric configuration resolving, in addition, subcompartments of G1. Quantitative restoration of protein fluorescence was achieved by introducing acidic EDTA washes to strip the copper from these proteins which were then imaged at neutral pH. The rate of synthesis of this Golgi membrane marker was low during early G1, but in the second half of G1 (30% of cycle duration) much of the synthesis occurred. Marker synthesis then persisted during S and G2. These insights into Golgi biology are discussed in terms of the cell's ability to adapt exocytosis to cell growth needs.

EmbRS a new two-component system that inhibits biofilm formation and saves Rubrivivax gelatinosus from sinking.

Photosynthetic bacteria can switch from planktonic lifestyle to phototrophic biofilm in mats in response to environmental changes. The mechanisms of phototrophic biofilm formation are, however, not characterized. Herein, we report a two-component system EmbRS that controls the biofilm formation in a photosynthetic member of the Burkholderiales order, the purple bacterium Rubrivivax gelatinosus. EmbRS inactivation results in cells that form conspicuous bacterial veils and fast-sinking aggregates in liquid. Biofilm analyses indicated that EmbRS represses the production of an extracellular matrix and biofilm formation. Mapping of transposon mutants that partially or completely restore the wild-type (WT) phenotype allowed the identification of two gene clusters involved in polysaccharide synthesis, one fully conserved only in Thauera sp., a floc-forming wastewater bacterium. A second two-component system BmfRS and a putative diguanylate cyclase BdcA were also identified in this screen suggesting their involvement in biofilm formation in this bacterium. The role of polysaccharides in sinking of microorganisms and organic matter, as well as the importance and the evolution of such regulatory system in phototrophic microorganisms are discussed.

Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation.

Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclin-dependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis (Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication, indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization. Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithorax-related protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required for cell elongation.

Distinct lytic vacuolar compartments are embedded inside the protein storage vacuole of dry and germinating Arabidopsis thaliana seeds.

Plant cell vacuoles are diverse and dynamic structures. In particular, during seed germination, the protein storage vacuoles are rapidly replaced by a central lytic vacuole enabling rapid elongation of embryo cells. In this study, we investigate the dynamic remodeling of vacuolar compartments during Arabidopsis seed germination using immunocytochemistry with antibodies against tonoplast intrinsic protein (TIP) isoforms as well as proteins involved in nutrient mobilization and vacuolar acidification. Our results confirm the existence of a lytic compartment embedded in the protein storage vacuole of dry seeds, decorated by γ-TIP, the vacuolar proton pumping pyrophosphatase (V-PPase) and the metal transporter NRAMP4. They further indicate that this compartment disappears after stratification. It is then replaced by a newly formed lytic compartment, labeled by γ-TIP and V-PPase but not AtNRAMP4, which occupies a larger volume as germination progresses. Altogether, our results indicate the successive occurrence of two different lytic compartments in the protein storage vacuoles of germinating Arabidopsis cells. We propose that the first one corresponds to globoids specialized in mineral storage and the second one is at the origin of the central lytic vacuole in these cells.

Assessment of mitochondria as a compartment for phosphatidylinositol synthesis in Solanum tuberosum.

The outer mitochondrial membrane is particularly rich in phosphatidylinositol (PtdIns), a phospholipid found in different amounts in all eukaryotic membranes, but not synthesized in situ by all. PtdIns is therefore subjected to traffic from the synthesizing membranes to the non-synthesizing ones. The contribution of mitochondria to the cell PtdIns pool has never been the focus of a specific study in plants, whereas in yeast, the presence of the enzyme responsible for synthesis, PtdIns synthase (PIS, cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11), has clearly been demonstrated in mitochondria. As these organelles have now been shown to be responsible for the synthesis of several lipids, the present work aimed at evaluating mitochondria as a compartment for the synthesis of PtdIns in plants. The sub-cellular localization of PIS was studied in Solanum tuberosum L. by membrane fractionation, enzymatic analysis and by confocal microscopy in living cells. In potato, beside the endoplasmic reticulum, the activity of PIS was found to be tightly associated to mitochondria. Using a fluorescent reporter fusion, the enzyme was also found to be associated to these organelles. The enzyme was not present at the plasma membrane. A comparison of the localization in other cell systems suggests that the mitochondrial localization could be regulated.

Exploring plant endomembrane dynamics using the photoconvertible protein Kaede.

Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack.

Toll-like receptor pathways in the immune responses to mycobacteria.

The control of Mycobacterium tuberculosis infection depends on recognition of the pathogen and the activation of both the innate and adaptive immune responses. Toll-like receptors (TLR) were shown to play a critical role in the recognition of several pathogens. Mycobacterial antigens recognise distinct TLR resulting in rapid activation of cells of the innate immune system. Recent evidence from in vitro and in vivo investigations, summarised in this review demonstrates TLR-dependent activation of innate immune response, while the induction of adaptive immunity to mycobacteria may be TLR independent.