Using an atomic force microscope, we have studied threedimensional molecular topography and calcium-sensitive conformational changes of individual hemichannels. Fulllength (non-truncated) Cx43 hemichannels (connexons), when reconstituted in lipid bilayer, appear as randomly distributed individual particles and clusters. They show a lack of preferential orientation of insertion into lipid membrane; in a single bilayer, connexons with protrusion of either the extracellular face or the large non-truncated cytoplasmic face are observed. Extracellular domains of these undocked hemichannels are structurally different from hemichannels in the docked gap junctional plaques examined after their exposure by force dissection or chemical dissection. Calcium induced a reversible change in the extracellular pore diameter. Hemichannels imaged in a physiological buffer with 1.8 mM Ca ؉2 had the pore diameter of ϳ1.8 nm, consistent with the closed channel conformation. Reducing Ca ؉2 concentration to ϳ1.4, 1, and 0 mM, which changes hemichannels from the closed to open conformation, increased the pore diameter to ϳ2.5 nm for ϳ27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca 2؉ ]-dependent. Computational analysis of the atomic force microscopy phase mode imaging reveals a significantly higher interfacial energy for open hemichannels that results from the interactions between the atomic force microscope probe and the hydrophobic domains. Thus, hydrophobic extracellular domains of connexins regulate calcium-dependent conformational changes.

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Atomic force microscopy (AFM) was used to image celery (Apium graveolens L.) parenchyma cell walls in situ. Cellulose microfibrils could clearly be distinguished in topographic images of the cell wall. The microfibrils of the hydrated walls appeared smaller, more uniformly distributed, and less enmeshed than those of dried peels. In material that was kept hydrated at all times and imaged under water, the microfibril diameter was mainly in the range 6-25 nm. The cellulose microfibril diameters were highly dependent on the water content of the specimen. As the water content was decreased, by mixing ethanol with the bathing solution, the microfibril diameters increased. Upon complete dehydration of the specimen we observed a significant increase in microfibril diameter. The procedure used to dehydrate the parenchyma cells also influenced the size of cellulose microfibrils with freeze-dried material having larger diameters than air-dried material.

The primary walls of celery (Apium graveolens L.) parenchyma cells were isolated and their polysaccharide components characterized by glycosyl linkage analysis, cross-polarization magic-angle spinning solid-state 13C nuclear magnetic resonance (CP/MAS 13C NMR) and X-ray diffraction. Glycosyl linkage analysis showed that the cell walls consisted of mainly cellulose (43 mol%) and pectic polysaccharides (51 mol%), comprising rhamnogalacturonan (28 mol%), arabinan (12 mol%) and galactan (11 mol%). The amounts of xyloglucan (2 mol%) and xylan (2 mol%) detected in the cell walls were strikingly low. The small amount of xyloglucan present means that it cannot coat the cellulose microfibrils. Solid-state 13C NMR signals were consistent with the constituents identified by glycosyl linkage analysis and allowed the walls to be divided into three domains, based on the rigidity of the polymers. Cellulose (rigid) and rhamnogalacturonan (semi-mobile) polymers responded to the CP/MAS 13C NMR pulse sequence and were distinguished by differences in proton spin relaxation time constants. The arabinans, the most mobile polymers, responded to single-pulse excitation (SPE), but not CP/MAS 13C NMR. From solid-state 13C NMR of the cell walls the diameter of the crystalline cellulose microfibrils was determined to be approximately 3 nm while X-ray diffraction of the cell walls gave a value for the diameter of approximately 2 nm.

The accumulation of abnormal tau filaments is a pathological hallmark of many neurodegenerative diseases. In 1998, genetic analyses revealed a direct linkage between structural and regulatory mutations in the tau gene and the neurodegenerative disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Importantly, the FTDP-17 phenotype is transmitted in a dominant rather than a recessive manner. However, the underlying molecular mechanisms causing disease remain uncertain. The most common molecular mechanism generating dominant phenotypes is the loss of function of a multimeric complex containing both mutant and wild-type subunits. Therefore, we sought to determine whether tau might normally function as a multimer. We co-incubated 35 S-radiolabeled tau and biotinylated tau with taxol stabilized microtubules, at very low molar ratios of tau to tubulin. Subsequent covalent cross-linking followed by affinity-precipitation of the biotinylated tau revealed the formation of microtubule-dependent tau oligomers. We next used atomic force microscopy to independently assess this conclusion. Our results are consistent with the hypothesis that tau forms oligomers upon binding to microtubules. In addition to providing insights into normal tau action, our findings lead us to propose that one mechanism by which mutations in tau may cause cell death is through the formation of tau complexes containing mutant tau molecules in association with wild-type tau. These wildtype-mutant tau complexes may possess altered biological and/or biophysical properties that promote onset of the FTDP-17 phenotype, including neuronal cell death by either altering normal tau-mediated regulation of microtubule-dependent cellular functions and/or promoting the formation of pathological tau aggregates.The microtubule-associated protein tau, localized predominantly in the cell bodies and axons of neuronal cells, is necessary for the establishment of neuronal cell polarity and axon outgrowth, for axonal transport, and the maintenance of axonal morphology (for example, see Refs. 1-6). Tau is also expressed in glial cells (7), although its role (8) there are less defined.Mechanistically, tau is well known to stimulate MT 1 polymerization, to stabilize MTs, and to modulate MT dynamics (9 -12). Since MT dynamics must be tightly regulated for cells to function and remain viable (e.g. see Refs. 13 and 14), it follows that tau action must also be finely regulated in cells.Alternatively, abnormal tau behavior is often associated with neurodegenerative diseases. In Alzheimer's disease, FTDP-17 and a large number of additional "tauopathies," necrotic neurons possess abnormal pathological fibers composed primarily of hyperphosphorylated and dysfunctional tau (for a recent review, see Ref. 15). Until recently, the relationship between tau and these various diseases was only correlative. However, in 1998, several groups reported a direct genetic linkage between mutations in the tau gene and FTDP-17, a group of related neurodegenerative con...