Background of Plant Vacuoles Patch-Clamp Lysosomal Electrophysiology
Plant cells initially formed by cytokinesis in the apical meristematic tissue of the stem or root contain hundreds of small protoplastic pores that arise from the outgrowth and fusion of vesicles in the trans-Golgi network. At this stage of development, plant vesicles are similar in size and number to animal lysosomes. However, as the plant cell expands, these tiny vesicles gradually fuse to form a central vesicle. Plant cells are unique in that they contain a large number of acidic vesicles that occupy most of the cell's volume. It contains most of the cellular complement K+, Ca2+, sugars, organic acids and other solutes, many of which must be actively transported to counter their electrochemical gradients. As a result, considerable energy must be expended to maintain the solute concentration within the central vesicle. Due to their simple isolation and large size, vesicles are well suited for patch clamp studies.
Fig. 1. Flowchart of experimental procedures showing how the Arabidopsis vacuole becomes a heterologous expression system for animal lysosomal ion channels and transporters. (Matzke AJ, et al., 2010)
Plant Vacuoles Patch-Clamp Lysosomal Electrophysiology Services
Because of the similarities in transport and targeting between vesicle membranes and lysosomal membrane proteins, we use plant vesicles as heterologous expression systems for lysosomal channels and transport proteins. At CD BioSciences, we are committed to providing customized patch-clamp lysosomal electrophysiology services for plant vesicles to our global customers by transforming protoplasts from Arabidopsis wild-type plants or mutants lacking specific endogenous channels or transporter proteins.
Our skilled scientists have successfully constructed a technical platform for delivery of the major channel and transporter protein families of lysosomal membranes to the vesicle membrane in plant cells. We combine fluorescent indicator dyes and membrane clamp technology to help our clients identify and characterize many types of endogenous lysosomal ion channels and transporter proteins. Here, we develop a customized procedure for analyzing plant vesicle lysosomal electrophysiology based on the membrane clamp technique.
(1) Arabidopsis plants are cultured in growth chambers under controlled light and temperature conditions, their leaf pulp protoplasts are isolated.
(2) Cloning of cDNAs of animal intracellular channels or transporter proteins into suitable plant expression vectors for high protein expression.
(3) Combine fluorescent marker fusions to verify protein expression and localization.
(4) Vesicles are released from transformed protoplasts for subsequent membrane clamp experiments.
(5) Perform membrane clamp measurements on plant vesicles in the following configurations.
- With a vesicle attached, allowing measurement of the current in the membrane portion directly below the pipette tip, but without control of the contents of the vesicle lumen.
- The whole vesicle, which allows macroscopic current recordings mediated by channels or transport proteins present throughout the vesicle membrane.
- Cytoplasmic lateral and vesicle lateral excision patches, which are used to detect single-channel currents.
Why Choose Us
- Channel regulation of cytosolic factors can be easily studied.
- The dedicated perfusion system can be exchanged at will during the experiment.
- Experienced team.
- Mature plasmid transformation technology platform.
CD BioSciences can meet any reasonable needs of our clients, taking time and budget into consideration for you. Our aim is to be customer-centric and to provide the highest quality services to customers. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.
Reference
- Festa M, et al. (2022) Current Methods to Unravel the Functional Properties of Lysosomal Ion Channels and Transporters. Cells.11(6):921.