Spectrin and Ankyrin-Based Pathways: Metazoan Inventions for Integrating Cells Into Tissues

2001 ◽  
Vol 81 (3) ◽  
pp. 1353-1392 ◽  
Author(s):  
Vann Bennett ◽  
Anthony J. Baines
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Striated Muscle ◽  
Membrane Skeleton ◽  
Tissue Level ◽  
Yeast Genome ◽  
Mitotic Spindles ◽  
Molecular Physiology ◽  
Mammalian Erythrocyte ◽  
Associated Proteins

The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca2+homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

scholarly journals Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

1997 ◽  
Vol 17 (9) ◽  
pp. 5210-5226 ◽  
Author(s):  
V I Titorenko ◽  
D M Ogrydziak ◽  
R A Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Plasma Membrane ◽  
Protein Secretion ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mycelial Form ◽  
Secretory Pathways ◽  
Associated Proteins ◽  
Yeast Yarrowia Lipolytica

We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum.

A comparison of membrane fracture faces of fixed and unfixed glycerinated tissue

1976 ◽  
Vol 21 (3) ◽  
pp. 437-448
Author(s):  
A.S. Breathnach ◽  
M. Gross ◽  
B. Martin ◽  
C. Stolinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Striated Muscle ◽  
Freeze Fracture ◽  
Particle Aggregation ◽  
Buccal Epithelium ◽  
Mitochondrial Membranes ◽  
Nuclear Membranes ◽  
General Plasma

Fixed (glutaraldehyde, 3%) and unfixed specimens of rat buccal epithelium, striated muscle, and liver, were cryoprotected with glycerol, freeze-fractured, and replicated without sublimation. A comparison of fracture faces of general plasma membranes, nuclear membranes, mitochondrial membranes, and membranes of rough endoplasmic reticulum revealed no significant differences as between fixed and unfixed material. Apart from some membranes of liver endoplasmic reticulum, there was no evidence of aggregation or redistribution of intramembranous particles in the unfixed material. The results demonstrate that chemical prefixation of tissues for freeze-fracture is not always necessary, or even desirable, and that glycerol may not be as deeply or directly implicated in particle aggregation as previously thought. Fixation with glutaraldehyde alters the cleaving behaviour of plasma membrane at desmosomes and tight junctions, but not at gap junctions.

scholarly journals Deciphering the Cellular Functions of the Op18/Stathmin Family of Microtubule-Regulators by Plasma Membrane-targeted Localization

2003 ◽  
Vol 14 (9) ◽  
pp. 3716-3729 ◽  
Author(s):  
Per Holmfeldt ◽  
Kristoffer Brännström ◽  
Sonja Stenmark ◽  
Martin Gullberg
Keyword(s):  
Plasma Membrane ◽  
Direct Interaction ◽  
Surface Protein ◽  
Ternary Complexes ◽  
Cell Surface Protein ◽  
Mitotic Spindles ◽  
Cellular Functions ◽  
Tubulin Binding ◽  
Associated Proteins ◽  
Cellular Compartments

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail–aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.

Synthesis and Turnover of Membrane Proteins in Rat Liver: An Examination of the Membrane Flow Hypothesis

1971 ◽  
Vol 26 (10) ◽  
pp. 1031-1039 ◽  
Author(s):  
Werner W. Franke ◽  
D. James Morre ◽  
Barbara Deumling ◽  
Ronald D. Cheetham ◽  
Jürgen Kartenbeck ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Golgi Apparatus ◽  
Plasma Membranes ◽  
Degradation Kinetics ◽  
Specific Radioactivity ◽  
Membrane Flow ◽  
Rapid Turnover ◽  
Associated Proteins

The kinetics of synthesis and degradation of the protein constituents of nuclear membranes, endoplasmic reticulum membranes (rough-surfaced microsomes), Golgi apparatus membranes and plasma membranes were determined following a single administration of L- [guanido-14C] arginine by intraperitoneal injection. Membrane protein was determined as the fraction which resists sonication and sequential extrations with 1.5 M KCl, 0.1% deoxycholate and water to remove intravesicular, intracisternal (secretory), nucleo-, adsorbed and ribosome-associated proteins.The order of maximum labeling of membrane proteins was a) endoplasmic reticulum (nuclear membrane), b) Golgi apparatus, and c) plasma membrane. Rapid decreases in specific radioactivity followed maximal labeling of endoplasmic reticulum and Golgi apparatus membranes. These rapid turnover components of endoplasmic reticulum and Golgi apparatus were sufficient to account for labeling of plasma membranes via a flow mechanism.Incorporation of radioactivity into plasma membranes showed two distinct phases. The ultrastructural features underlying the biphasic pattern of incorporation into plasma membranes are discussed.Following initial incorporation and rapid turnover, membrane proteins were characterized by degradation kinetics approximating 1st order. Rates of degradation for Golgi apparatus and plasma membranes were faster than those for nuclear envelope and endoplasmic reticulum membranes.Assuming steady state conditions, an absolute synthetic rate of 7.1 mpg/min/avergage hepatocyte was calculated for membrane proteins of the plasma membrane.The results are compatible with intracellular movement and conversion of rough endoplasmic reticulum to plasma membrane via the membranes of the Golgi apparatus, i. e., membrane flow. Additionally, the kinetics indicate that membrane synthesis and transfer is restricted to specific parts of the endoplasmic reticulum and Golgi apparatus.

Tropomodulins and tropomyosins – organizers of cellular microcompartments

2013 ◽  
Vol 4 (1) ◽  
pp. 89-101 ◽  
Author(s):  
Thomas Fath
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Critical Role ◽  
Eukaryotic Cells ◽  
Multigene Families ◽  
Cellular Functions ◽  
Cellular Morphogenesis ◽  
Wide Range ◽  
Associated Proteins

AbstractEukaryotic cells show a remarkable compartmentalization into compartments such as the cell nucleus, the Golgi apparatus, the endoplasmic reticulum, and endosomes. However, organelle structures are not the only means by which specialized compartments are formed. Recent research shows a critical role for diverse actin filament populations in defining functional compartments, here referred to as microcompartments, in a wide range of cells. These microcompartments are involved in regulating fundamental cellular functions including cell motility, plasma membrane organization, and cellular morphogenesis. In this overview, the importance of two multigene families of actin-associated proteins, tropomodulins and tropomyosins, their interactions with each other, and a large number of other proteins will be discussed in the context of generating specialized actin-based microcompartments.

scholarly journals The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver

1994 ◽  
Vol 300 (2) ◽  
pp. 419-427 ◽  
Author(s):  
J P Lièvremont ◽  
A M Hill ◽  
M Hilly ◽  
J P Mauger
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Alkaline Treatment ◽  
Inositol 1,4,5 Trisphosphate ◽  
Associated Proteins ◽  
Bile Canalicular Membrane ◽  
Low Levels

Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

scholarly journals Supramolecular architecture of endoplasmic reticulum–plasma membrane contact sites

2016 ◽  
Vol 44 (2) ◽  
pp. 534-540 ◽  
Author(s):  
Rubén Fernández-Busnadiego
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Striated Muscle ◽  
Electron Tomography ◽  
Structural Diversity ◽  
Molecular Organization ◽  
Supramolecular Architecture ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact

The endoplasmic reticulum (ER) forms membrane contact sites (MCS) with most other cellular organelles and the plasma membrane (PM). These ER–PM MCS, where the membranes of the ER and PM are closely apposed, were discovered in the early days of electron microscopy (EM), but only recently are we starting to understand their functional and structural diversity. ER–PM MCS are nowadays known to mediate excitation–contraction coupling (ECC) in striated muscle cells and to play crucial roles in Ca2+ and lipid homoeostasis in all metazoan cells. A common feature across ER–PM MCS specialized in different functions is the preponderance of cooperative phenomena that result in the formation of large supramolecular assemblies. Therefore, characterizing the supramolecular architecture of ER–PM MCS is critical to understand their mechanisms of function. Cryo-electron tomography (cryo-ET) is a powerful EM technique uniquely positioned to address this issue, as it allows 3D imaging of fully hydrated, unstained cellular structures at molecular resolution. In this review I summarize our current structural knowledge on the molecular organization of ER–PM MCS and its functional implications, with special emphasis on the emerging contributions of cryo-ET.

Triple Fixation For Electron Microscopy

1968 ◽  
Vol 26 ◽  
pp. 90-91 ◽  
Author(s):  
M. A. Hayat
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Electron Beam ◽  
Plasma Membrane ◽  
Myelin Sheath ◽  
Good Deal ◽  
Granular Structure ◽  
Oxidizing Agent ◽  
Fixation Technique ◽  
Large Clusters

Potassium permanganate has been successfully employed to study membranous structures such as endoplasmic reticulum, Golgi, plastids, plasma membrane and myelin sheath. Since KMnO4 is a strong oxidizing agent, deposition of manganese or its oxides account for some of the observed contrast in the lipoprotein membranes, but a good deal of it is due to the removal of background proteins either by dehydration agents or by volatalization under the electron beam. Tissues fixed with KMnO4 exhibit somewhat granular structure because of the deposition of large clusters of stain molecules. The gross arrangement of membranes can also be modified. Since the aim of a good fixation technique is to preserve satisfactorily the cell as a whole and not the best preservation of only a small part of it, a combination of a mixture of glutaraldehyde and acrolein to obtain general preservation and KMnO4 to enhance contrast was employed to fix plant embryos, green algae and fungi.

The Advantages of Cryotechniques: Application To Bioluminescent Cells

1990 ◽  
Vol 48 (1) ◽  
pp. 486-487
Author(s):  
Gisèle Nicolas ◽  
Jean-Marie Bassot ◽  
Marie-Thérèse Nicolas
Keyword(s):  
Endoplasmic Reticulum ◽  
Striated Muscle ◽  
Light Emission ◽  
Stable State ◽  
Freeze Substitution ◽  
Initial Step ◽  
Point Of Interest ◽  
Cell Components ◽  
Excellent Preservation ◽  
External Water

The use of fast-freeze fixation (FFF) followed by freeze-substitution (FS) brings substantial advantages which are due to the extreme rapidity of this fixation compared to the conventional one. The initial step, FFF, physically immobilizes most molecules and therefore arrests the biological reactions in a matter of milliseconds. The second step, FS, slowly removes the water content still in solid state and, at the same time, chemically fixes the other cell components in absence of external water. This procedure results in an excellent preservation of the ultrastructure, avoids osmotic artifacts,maintains in situ most soluble substances and keeps up a number of cell activities including antigenicities. Another point of interest is that the rapidity of the initial immobilization enables the capture of unstable structures which, otherwise, would slip towards a more stable state. When combined with electrophysiology, this technique arrests the ultrastructural modifications at a well defined state, allowing a precise timing of the events.We studied the epithelium of the elytra of the scale-worm, Harmothoe lunulata which has excitable, conductible and bioluminescent properties. The intracellular sites of the light emission are paracrystals of endoplasmic reticulum (PER), named photosomes (Fig.1). They are able to flash only when they are coupled with plasma membrane infoldings by dyadic or triadic junctions (Fig.2) basically similar to those of the striated muscle fibers. We have studied them before, during and after stimulation. FFF-FS showed that these complexes are labile structures able to diffentiate and dedifferentiate within milliseconds. Moreover, a transient network of endoplasmic reticulum was captured which we have named intermediate endoplasmic reticulum (IER) surrounding the PER (Fig.1). Numerous gap junctions are found in the membranous infoldings of the junctional complexes (Fig.3). When cryofractured, they cleave unusually (Fig.4-5). It is tempting to suggest that they play an important role in the conduction of the excitation.

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