MinE recruits, stabilizes, releases, and inhibits MinD interactions with membrane to drive oscillation

SUMMARYThe MinD and MinE proteins ofEscherichia coliself-organize into a standing-wave oscillator on the membrane to help align division at mid-cell. When unleashed from cellular confines, we find that MinD and MinE form a wide spectrum of patterns on artificial bilayers - static amoebas, traveling waves, traveling mushrooms, and bursts with standing-wave dynamics. We recently focused our cell-free studies on bursts because their dynamics closely resemble those foundin vivo. The data unveiled a patterning mechanism largely governed by MinE regulation of MinD interaction with membrane. We proposed that the MinD to MinE ratio on the membrane acts as a toggle switch between MinE-stimulated recruitment or release of MinD from the membrane. Here we provide data that further refines and extends our model that explains the remarkable spectrum of patterns supported by these two ‘simple’ proteins.

The influence of sterols on the conformation of recombinant mitochondrial porin in detergent

Mitochondrial porins (voltage-dependent anion-selective channels, VDAC) are key contributors to cellular metabolism. When isolated from mitochondria porins copurify with sterols, and some isolated forms of the protein require sterol for insertion into artificial membranes. Nonetheless, the contributions of sterols to the folded state of mitochondrial porin are not understood. Recently, with the goal of high-resolution structural studies, several laboratories have developed methods for folding recombinant porins at high concentration in detergent. In the present study, recombinant Neurospora crassa porin solubilized in detergent–sterol mixtures was examined. Sterols do not significantly alter the secondary structure of porin in lauryl dimethylamine oxide, nor in a mixture of sodium dodecylsulfate and dodecylmaltopyranoside. However, as detected by near-UV circular dichroism spectropolarimetry and fluorescence spectroscopy, the environments surrounding the aromatic amino acids in the detergent–sterol solubilized protein are measurably different from those in detergent alone. Furthermore, the effects are different in the presence of ergosterol, the native sterol in fungal mitochondria, and cholesterol. While these influences on the tertiary arrangement of detergent-solubilized porin are subtle, they may contribute to the generation of a form of the protein competent for insertion into the artificial bilayers used for electrophysiological analyses, and should be considered in future structural studies of porin.

Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.

The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

Phospholipids undergo hop diffusion in compartmentalized cell membrane

The diffusion rate of lipids in the cell membrane is reduced by a factor of 5–100 from that in artificial bilayers. This slowing mechanism has puzzled cell biologists for the last 25 yr. Here we address this issue by studying the movement of unsaturated phospholipids in rat kidney fibroblasts at the single molecule level at the temporal resolution of 25 μs. The cell membrane was found to be compartmentalized: phospholipids are confined within 230-nm-diameter (ϕ) compartments for 11 ms on average before hopping to adjacent compartments. These 230-nm compartments exist within greater 750-nm-ϕ compartments where these phospholipids are confined for 0.33 s on average. The diffusion rate within 230-nm compartments is 5.4 μm2/s, which is nearly as fast as that in large unilamellar vesicles, indicating that the diffusion in the cell membrane is reduced not because diffusion per se is slow, but because the cell membrane is compartmentalized with regard to lateral diffusion of phospholipids. Such compartmentalization depends on the actin-based membrane skeleton, but not on the extracellular matrix, extracellular domains of membrane proteins, or cholesterol-enriched rafts. We propose that various transmembrane proteins anchored to the actin-based membrane skeleton meshwork act as rows of pickets that temporarily confine phospholipids.

The Power of Freeze-Fracture Technique in Electron Microscopy

In the early 1960s, concerns about artifacts in preparing biological material for electron microscopy led to a new technique whereby samples are rapidly frozen, fractured under high vacuum, the fractured surfaces shadowed and replicated with a thin metal-carbon coat, and the cleaned replica examined in a transmission electron microscope. Pioneered by Moor and Muhlethaler subcellular structures are revealed with extraordinary three-dimensional clarity at near-molecular resolution. Furthermore, it was observed and proven at model systems as well as biological membranes that lipid bilayers split along their hydrophobic interior during freeze-fracture procedure. Therefore, freeze-fracture electron microscopy (FFEM) has the unique advantage of accessing the hydrophobic interior of biological (FIG.l, 5 and 6) as well as artificial bilayers (FIG. 2, 4, 5, and 6). Here it permits study of pattern generated by intrinsic proteins as well as lipids (FIG. 1 and 2).

The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.