Flow cytometric analysis and sorting of plant mitotic chromosomes has been mastered by only a few laboratories worldwide. Yet, it has been contributing significantly to progress in plant genetics, including the production of genome assemblies and the cloning of important genes. The dissection of complex genomes by flow sorting into the individual chromosomes that represent small parts of the genome reduces DNA sample complexity and streamlines projects relying on molecular and genomic techniques. Whereas flow cytometric analysis, that is, chromosome classification according to fluorescence and light scatter properties, is an integral part of any chromosome sorting project, it has rarely been used on its own due to lower resolution and sensitivity as compared to other cytogenetic methods. To perform chromosome analysis and sorting, commercially available electrostatic droplet sorters are suitable. However, in order to resolve and purify chromosomes of interest the instrument must offer high resolution of optical signals as well as stability during long runs. The challenge is thus not the instrumentation, but the adequate sample preparation. The sample must be a suspension of intact mitotic metaphase chromosomes and the protocol, which includes the induction of cell cycle synchrony, accumulation of dividing cells at metaphase, and release of undamaged chromosomes, is time consuming and laborious and needs to be performed very carefully. Moreover, in addition to fluorescent staining chromosomal DNA, the protocol may include specific labelling of DNA repeats to facilitate discrimination of particular chromosomes. This review introduces the applications of chromosome sorting in plants, and discusses in detail sample preparation, chromosome analysis and sorting to achieve the highest purity in flow-sorted fractions, and their suitability for downstream applications.
Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.
- MeSH
- Cell Cycle MeSH
- Chromosomes, Plant MeSH
- Cytogenetics MeSH
- DNA, Plant genetics MeSH
- In Situ Hybridization, Fluorescence methods MeSH
- Karyotyping MeSH
- Meristem cytology MeSH
- Flow Cytometry methods MeSH
- Plant Cells MeSH
- Plants genetics MeSH
- Seeds growth & development MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
- MeSH
- Cell Line MeSH
- Hyaluronoglucosaminidase MeSH
- Mice MeSH
- Transplantation Immunology MeSH
- Check Tag
- Mice MeSH
Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.
- MeSH
- Chromosomes, Plant ultrastructure MeSH
- Genome Size MeSH
- Microscopy, Fluorescence MeSH
- Genome, Plant * MeSH
- In Situ Hybridization, Fluorescence MeSH
- Hordeum cytology genetics MeSH
- Germination genetics MeSH
- Metaphase genetics MeSH
- Polyploidy MeSH
- Flow Cytometry methods MeSH
- Triticum cytology genetics MeSH
- Sequence Analysis, DNA MeSH
- Seeds genetics MeSH
- Chromosomes, Artificial, Bacterial MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Secale cytology genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.
- MeSH
- Adaptor Proteins, Signal Transducing chemistry metabolism MeSH
- Cell Membrane metabolism MeSH
- Extracellular Space chemistry MeSH
- Glycosylation MeSH
- HEK293 Cells MeSH
- HeLa Cells MeSH
- Jurkat Cells MeSH
- Humans MeSH
- Lipoylation * MeSH
- Membrane Proteins chemistry metabolism MeSH
- Protein Structure, Tertiary MeSH
- Protein Transport MeSH
- Structure-Activity Relationship MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Despite the development of several cultivation methods, the rate of discovery of microorganisms that are yet-to-be cultivated outpaces the rate of isolating and cultivating novel species in the laboratory. Furthermore, no current cultivation technique is capable of selectively isolating and cultivating specific bacterial taxa or phylogenetic groups independently of morphological or physiological properties. Here, we developed a new method to isolate living bacteria solely based on their 16S rRNA gene sequence. We showed that bacteria can survive a modified version of the standard fluorescence in situ hybridization (FISH) procedure, in which fixation is omitted and other factors, such as centrifugation and buffers, are optimized. We also demonstrated that labelled DNA probes can be introduced into living bacterial cells by means of chemical transformation and that specific hybridization occurs. This new method, which we call live-FISH, was then combined with fluorescence-activated cell sorting (FACS) to sort specific taxonomic groups of bacteria from a mock and natural bacterial communities and subsequently culture them. Live-FISH represents the first attempt to systematically optimize conditions known to affect cell viability during FISH and then to sort bacterial cells surviving the procedure. No sophisticated probe design is required, making live-FISH a straightforward method to be potentially used in combination with other single-cell techniques and for the isolation and cultivation of new microorganisms.
- MeSH
- Bacillus genetics MeSH
- Bacteria genetics MeSH
- RNA, Bacterial genetics MeSH
- DNA Probes MeSH
- Phylogeny MeSH
- In Situ Hybridization, Fluorescence * MeSH
- Microbiological Techniques * MeSH
- Flow Cytometry MeSH
- RNA, Ribosomal, 16S genetics MeSH
- Cell Separation * MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- MeSH
- Leukemia, Myeloid, Acute diagnosis etiology genetics MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell diagnosis etiology genetics MeSH
- Hematologic Neoplasms * diagnosis etiology genetics MeSH
- Hematologic Tests * methods utilization MeSH
- Clinical Laboratory Techniques methods utilization MeSH
- Clinical Trials as Topic MeSH
- Humans MeSH
- Polymerase Chain Reaction methods utilization MeSH
- Flow Cytometry methods utilization MeSH
- Neoplasm, Residual * diagnosis MeSH
- Blood Component Removal methods utilization MeSH
- Check Tag
- Humans MeSH
Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.
- MeSH
- Cell Membrane metabolism MeSH
- Chlorocebus aethiops MeSH
- COS Cells MeSH
- Endosomes metabolism MeSH
- Phosphotransferases (Alcohol Group Acceptor) metabolism MeSH
- Membrane Fusion physiology MeSH
- Humans MeSH
- Vesicle-Associated Membrane Protein 3 metabolism MeSH
- SNARE Proteins metabolism MeSH
- Receptors, Transferrin metabolism MeSH
- Protein Transport physiology MeSH
- Vesicular Transport Proteins metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Intramural MeSH
