The Plasma Membrane of the Spermatozoon
Sek Wen Hui in Freeze-Fracture Studies of Membranes, 1989
Immediately below the zone where such basal cords are found is the posterior ring (Figures 7 and 8), a uniform belt approximately 40 nm in thickness extending around the entire circumference of the head-neck junction. This structure is invariably found in mammalian sperm and appears to be virtually identical in detailed structure among species. Included among these details is a highly ordered periodicity of 8 nm perpendicular to the belt direction (Figures 9 and 10). The analog of this structure in thin sections can easily be detected as a dense plaque sandwiched between the plasma and nuclear membranes (Figure 11). It is apparent that this structure may provide a “tight junction”-like seal between the cytosolic compartments of the sperm head and tail. Although there is no direct evidence of such a physiological seal, the differences in metabolic requirements between these compartments may make such a seal a highly useful if not mandatory component (see also Koehler12). Further suggestive evidence comes from recent studies using fluorescence recovery after photobleaching. Wolf et al.13 have shown that lipid diffusion is two to three times as rapid in the flagellar membrane of mouse sperm compared to that of the head.
Beyond Enzyme Kinetics
Clive R. Bagshaw in Biomolecular Kinetics, 2017
Photobleaching of fluorophores, which is often a technical limitation for following reactions in cells, can be exploited in the technique of fluorescence recovery after photobleaching (FRAP) [297–299]. The dynamic nature of cellular structures can be established by bleaching fluorophores in a small (micrometer) region using a focused laser beam and following the recovery as molecules outside the bleached region diffuse back into the bleached region to replenish the original molecules (Figure 5.15). Recovery of fluorescence by diffusion of individual proteins can occur on the millisecond time scale, consistent with their known diffusion constants (Table 3.1). However, recovery times are usually slower than in aqueous solution because protein must navigate through a web of cytoskeletal elements. Perhaps, more surprising is the recovery of many protein assemblies that involve not only diffusion but also the exchange of subunits on the seconds to minutes time scale and that demonstrate such complexes are highly dynamic. These include molecular motors, such as the bacterial flagellum, whose components are in continuous exchange while they operate [300]. These measurements demonstrate that self-assembly constitutes a series of reversible reactions and the resultant nanomachines are distinct from man-made macroscopic machines. We could not replace a damaged piston ring while a car is firing on the other three cylinders. FRAP has been used to elucidate the kinetic mechanism of transcription by RNA polymerases in vivo [301,302]. However, care is required in these studies because of reversible photobleaching of the fluorophores, particularly those involving green fluorescence protein (GFP) variants [303,304], may give rise to apparent recovery.
Dynamic Aspects of Cell Membrane Structure
Lelio G. Colombetti in Biological Transport of Radiotracers, 2020
A recent publication on the structure of cell membranes focused primarily on concepts regarding the organization of membrane components.1 The aim of the present article is to discuss the dynamics of membrane constituents, and to describe some of the consequences of their mobility on membrane function. As reviewed in the previous paper, early evidence revealed lateral mobility of both proteins and lipids in the plane of cell membranes.1 It was also shown that the catalytic function of certain membrane-bound enzymes is dependent on their interaction with surrounding lipid. Subsequently, processes restricting the lateral diffusion of intrinsic membrane proteins were described, e.g., the interaction between membrane spanning and cytoplasmic proteins such as glycophorin and spectrin in erythrocytes.1,2 Recent research on the mobility of membrane components has been markedly facilitated by the introduction of the method of fluorescence recovery after photobleaching (FRAP). The procedure is based on the complete destruction of photosensitive, fluorescent tags on proteins in a small, discrete area of the membrane upon exposure to a highly focused laser beam. Recovery of specific fluorescence in the area of the bleached spot reflects lateral migration of other molecules of the protein back into the area under study. By applying this technique, the mobility of membrane proteins has been described in various cell lines.3,4 In addition to supporting the general fluid-mosaic model of cell membranes, the results of these studies revealed that both the rate of protein migration and the amount returning were lower than theoretically expected.5 The finding that the path of return was longer than expected for randomly “floating” molecules suggests the existence in the lipid bilayer of areas of exclusion due to structural organization and/or interaction between neighboring molecules of proteins and/or lipids. The results, in fact, imply that some fraction of the protein population has been immobilized, apparently by interaction with cytoskeletal structures underlying the plasma membrane.
TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins
Published in Journal of Receptors and Signal Transduction, 2018
Radka Moravcova, Barbora Melkes, Jiri Novotny
The diffusion of proteins can be monitored by fluorescence recovery after photobleaching (FRAP). However, so far only a few studies have dealt with analysis of GPCR movement using this approach. Barak et al. [4] investigated β2-adrenergic receptor diffusibility on live cell membranes and demonstrated that real-time optical measurements of receptor interactions and dynamics on living cells are feasible. By using FRAP, Pucadyil et al. [5] found that the cell surface dynamics of serotonin1A receptor is modulated in a G protein-dependent manner. Besides that, it has been reported that μ-opioid receptor diffusion can be regulated by heterologous activation of other GPCRs, namely by α2-adrenergic or NPFF2 receptors [6]. There is also some evidence to suggest that agonists may affect receptor movement in the plasma membrane [7,8]. We have recently shown that the lateral mobility of µ-opioid receptor can be diversely influenced by biased agonists [9].
Tight junctions: from molecules to gastrointestinal diseases
Published in Tissue Barriers, 2023
Aekkacha Moonwiriyakit, Nutthapoom Pathomthongtaweechai, Peter R. Steinhagen, Papasara Chantawichitwong, Wilasinee Satianrapapong, Pawin Pongkorpsakol
As proteins related to the structure of the cell, the integrity of tight junctions was thought to provide a static, impermeable barrier at the site of apical intercellular space complexes. However, this hypothesis was proven to be almost entirely incorrect. In fact, the concept of tight junction dynamics has a long history of over 30 years.126 In the last decade, fluorescence recovery after photobleaching (FRAP) revealed different mobile characteristics of fluorescent protein-tagged tight junctions during a steady state.127–129 Although claudin-1 stably localized at the tight junction region, ZO-1 and occludin are differentially exchangeable at subcellular levels. Notably, tight junction-to-cytoplasm switching of ZO-1 was found to occur in an energy-dependent manner, which required activity of the actin-binding region (ABR) and MLCK. Indeed, ZO-1 switching could occur via either MLCK-dependent or MLCK-independent mechanisms, which are slow and fast kinetics, respectively.23 Meanwhile, occludin passively diffuses between apical and lateral membrane.23 Therefore, it is widely accepted that tight junction remodeling can spontaneously occur.127 These kinetic behaviors of three representative tight junction proteins refute the theory of tight junctions having a static architecture (Figure 3a).
Fluorescent probes for G-protein-coupled receptor drug discovery
Published in Expert Opinion on Drug Discovery, 2018
Christos Iliopoulos-Tsoutsouvas, Rohit N. Kulkarni, Alexandros Makriyannis, Spyros P. Nikas
Fluorescent-based imaging technologies are rapidly developing and can be used to ‘illuminate’ the pharmacology surrounding the ligand–GPCR interactions. Such technologies include Fluorescence Correlation Spectroscopy (FCS), Confocal Laser Scanning Microscopy (CLSM), Fluorescence Resonance Energy Transfer (FRET), Fluorescent Recovery After Photobleaching (FRAP), Fluorescence Polarization (FP) and super-resolution microscopy. Conventional light microscopy is a valuable tool, but its ability to resolve microscopic structures in optically thick specimens is limited. The invention of one-photon excitation confocal microscopy and two-photon excitation microscopy has begun to address 3D imaging needs in cells and tissues. Two-photon fluorescence microscopy allows three-dimensional imaging of biological specimens in vivo. Compared with confocal microscopy, it offers the advantages of increased tissue penetration, improved cell viability, reduced photo-bleaching and less autofluorescence but has the disadvantage of slightly lower resolution.
Related Knowledge Centers
- Diffusion
- Fluorescence
- Phospholipid
- Photobleaching
- Cell Membrane
- Molecular Binding
- Mercury
- Xenon
- Laser
- Diffusion Equation