Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using unfixed nuclear envelopes. ATP (1 mM) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing ATP analogue, ATP-gamma-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent a nuclear pore "contraction."
Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure-function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still nuclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.
Plasma membrane proteins are supposed to form clusters that allow 'functional cross-talk' between individual molecules within nanometre distance. However, such hypothetical protein clusters have not yet been shown directly in native plasma membranes. Therefore, we developed a technique to get access to the inner face of the plasma membrane of cultured transformed kidney (MDCK) cells. The authors applied atomic force microscopy (AFM) to visualize clusters of native proteins protruding from the cytoplasmic membrane surface. We used the K+ channel blocker iberiotoxin (IBTX), a positively charged toxin molecule, that binds with high affinity to plasma membrane potassium channels and to atomically flat mica. Thus, apical plasma membranes could be 'glued' with IBTX to the mica surface with the cytosolic side of the membrane accessible to the scanning AFM tip. The topography of these native inside-out membrane patches was imaged with AFM in electrolyte solution mimicking the cytosol. The plasma membrane could be clearly identified as a lipid bilayer with the characteristic height of 4.9 +/- 0.02 nm. Multiple proteins protruded from the lipid bilayer into the cytosolic space with molecule heights between 1 and 20 nm. Large protrusions were most likely protein clusters. Addition of the proteolytic enzyme pronase to the bath solution led to the disappearance of the proteins within minutes. The metabolic substrate ATP induced a shape-change of the protein clusters and smaller subunits became visible. ADP or the non-hydrolysable ATP analogue, ATP-gamma-S, could not exert similar effects. It is concluded that plasma membrane proteins (and/or membrane associated proteins) form 'functional clusters' in their native environment. The 'physiological' arrangement of the protein molecules within a cluster requires ATP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.