A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells (Allele 4.1 Madrid): Implications Regarding a Critical Role of Protein 4.1 in Maintenance of the Integrity of the Red Blood Cell Membrane

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG → AGG) of the protein 4.1 gene [the 4.1 (−) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 ± 63/μm2; normal, 504 ± 36/μm2). The number of basic units in the skeletal network was strikingly reduced (131 ± 21/μm2; normal, 548 ± 39/μm2), with decreased small-sized units (17 ± 4/μm2; normal, 384 ± 52/μm2) and increased large-sized units (64% ± 14%; normal, 5% ± 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.

A Markedly Disrupted Skeletal Network With Abnormally Distributed Intramembrane Particles in Complete Protein 4.1-Deficient Red Blood Cells (Allele 4.1 Madrid): Implications Regarding a Critical Role of Protein 4.1 in Maintenance of the Integrity of the Red Blood Cell Membrane

Electron microscopic (EM) studies were performed to clarify the interactions of membrane proteins in the red blood cell membrane structure in situ of a homozygous patient with total deficiency of protein 4.1 who carried a point mutation of the downstream translation initiation codon (AUG → AGG) of the protein 4.1 gene [the 4.1 (−) Madrid; Dalla Venezia et al, J Clin Invest 90:1713, 1992]. Immunologically, as expected, protein 4.1 was completely missing in the red blood cell membrane structure in situ. A markedly disrupted skeletal network was observed by EM using the quick-freeze deep-etching method and the surface replica method, although the number of spectrin molecules was only minimally reduced (395 ± 63/μm2; normal, 504 ± 36/μm2). The number of basic units in the skeletal network was strikingly reduced (131 ± 21/μm2; normal, 548 ± 39/μm2), with decreased small-sized units (17 ± 4/μm2; normal, 384 ± 52/μm2) and increased large-sized units (64% ± 14%; normal, 5% ± 1%). Concomitantly, immuno-EM disclosed striking clustering of spectrin molecules with aggregated ankyrin molecules in the red blood cell membrane structure in situ. Although no quantitative abnormalities in the number and size distribution of the intramembrane particles were observed, there was a disappearance of regular distribution, with many clusters of various sizes, probably reflecting the distorted skeletal network. Therefore, protein 4.1 suggests by EM to play a crucial role in maintenance of the normal integrity of the membrane structure in situ not only of the skeletal network but also of the integral proteins.

Freeze-fracture views of membrane motors of bacteria

The locomotory organelles of many bacteria are long, helical filaments called flagella. Bacterial flagella rotate. Flagellar rotation is driven by a molecular motor that is part of the membrane embedded flagellar base and fueled by electrochemical proton (or sodium) gradients. Rapid-freeze electron microscopy has revealed that rings of intramembrane particles found clustered around flagellar bases are a key structural module of this motor.The morphology of the ring particles provides clues to their function. The particles are located in the cytoplasmic membrane across which the electrochemical gradients that energize flagellar rotation are maintained. In Escherichia coli, in addition to proteins needed for assembly of the flagellum, two proteins, MotA and MotB, are required for motility. MotA conducts protons across membranes and interacts with MotB, a protein with a large periplasmic domain. In non-motile mutants lacking MotA and MotB, flagellar bases lack the particle rings. Plasmid based expression of both MotA and MotB is necessary for restoration of motility and the ring structures.