BRCA1/2 testing: uptake, phenocopies, and strategies to improve detection rates in initially negative families.

In families with clustering of breast and ovarian cancer, molecular testing of the major susceptibility genes BRCA1/2 helps to identify patients with disease mutations and healthy persons at high risk who can participate in targeted intervention programs. We investigated 5559 families from the German Consortium for Hereditary Breast and Ovarian Cancer included between 1997 and 2008 and treated under clinical routine conditions. In each family an index patient/person had been screened for deleterious mutations in BRCA1/2. Healthy relatives agreed to predictive testing in 888 of 1520 BRCA1/2 mutation-positive families (58%). Of 2646 eligible unaffected first-degree relatives 1143 decided to be tested (43%). In 325 families with BRCA1/2-positive index patients one related BC/OC patient was tested and 39 (12.0%; 95% confidence interval: 8.7-16.0%) discrepant cases found. A second related individual was screened in 163 of 3388 (4.9%) families with BRCA1/2-negative index patient and in eight families a BRCA1/2 mutation was found. In BRCA1/2 mutation-positive families, BC/OC patients lacking the familial mutation have to be expected at a rather high rate. In families with BRCA1/2-negative index patient we recommend a second screening if another patient with a high probability of carrying a BRCA1/2 mutation is available.

Signature polymorphisms in the internal transcribed spacer region relevant for the differentiation of zoophilic and anthropophilic strains of Trichophyton interdigitale and other species of T. mentagrophytes sensu lato.

BACKGROUND: Dermatophytes are the main cause of superficial mycoses in humans and animals. Molecular research has given useful insights into the phylogeny and taxonomy of the dermatophytes to overcome the difficulties with conventional diagnostics. OBJECTIVES: The Trichophyton mentagrophytes complex consists of anthropophilic as well as zoophilic species. Although several molecular markers have been developed for the differentiation of strains belonging to T. mentagrophytes sensu lato, correct identification still remains problematic, especially concerning the delineation of anthropophilic and zoophilic strains of T. interdigitale. This differentiation is not academic but is essential for selection of the correct antimycotic therapy to treat infected patients. METHODS: One hundred and thirty isolates identified by morphological characteristics as T. mentagrophytes sensu lato were investigated using restriction fragment length polymorphism (RFLP) and sequence analysis of the polymerase chain reaction-amplified internal transcribed spacer (ITS) region of the rDNA. RESULTS: Species of this complex produced individual RFLP patterns obtained by the restriction enzyme MvaI. Subsequent sequence analysis of the ITS1, 5.8S and ITS2 region of all strains, but of T. interdigitale in particular, revealed single unique polymorphisms in anthropophilic and zoophilic strains. CONCLUSIONS: Signature polymorphisms were observed to be useful for the differentiation of these strains and epidemiological data showed a host specificity among zoophilic strains of T. interdigitale/Arthroderma vanbreuseghemii compared with A. benhamiae as well as characteristic clinical pictures in humans when caused by zoophilic or anthropophilic strains. The delineation is relevant because it helps in determining the correct treatment and provides clues regarding the source of the infection.

Measurements of growth cone adhesion to culture surfaces by micromanipulation.

Neurons were grown on plastic surfaces that were untreated, or treated with polylysine, laminin, or L1 and their growth cones were detached from their culture surface by applying known forces with calibrated glass needles. This detachment force was taken as a measure of the force of adhesion of the growth cone. We find that on all surfaces, lamellipodial growth cones require significantly greater detachment force than filopodial growth cones, but this differences is, in general, due to the greater area of lamellipodial growth cones compared to filopodial growth cones. That is, the stress (force/unit area) required for detachment was similar for growth cones of lamellipodial and filopodial morphology on all surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces, which had a significantly lower stress of detachment than on other surfaces. Surprisingly, the forces required for detachment (760-3,340 mudynes) were three to 15 times greater than the typical resting axonal tension, the force exerted by advancing growth cones, or the forces of retraction previously measured by essentially the same method. Nor did we observe significant differences in detachment force among growth cones of similar morphology on different culture surfaces, with the exception of lamellipodial growth cones on L1-treated surfaces. These data argue against the differential adhesion mechanism for growth cone guidance preferences in culture. Our micromanipulations revealed that the most mechanically resistant regions of growth cone attachment were confined to quite small regions typically located at the ends of filopodia and lamellipodia. Detached growth cones remained connected to the substratum at these regions by highly elastic retraction fibers. The closeness of contact of growth cones to the substratum as revealed by interference reflection microscopy (IRM) did not correlate with our mechanical measurements of adhesion, suggesting that IRM cannot be used as a reliable estimator of growth cone adhesion.

Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites.

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5 degrees C for 2 h) or nocodazole (0.1 microgram/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 micron/min with no delay times other than the times to reach 37 degrees C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 microgram/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 microgram/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 micrograms/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 microgram/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly.(ABSTRACT TRUNCATED AT 400 WORDS)

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies.

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.

Growth cone behavior and production of traction force.

The growth cone must push its substrate rearward via some traction force in order to propel itself forward. To determine which growth cone behaviors produce traction force, we observed chick sensory growth cones under conditions in which force production was accommodated by movement of obstacles in the environment, namely, neurites of other sensory neurons or glass fibers. The movements of these obstacles occurred via three, different, stereotyped growth cone behaviors: (a) filopodial contractions, (b) smooth rearward movement on the dorsal surface of the growth cone, and (c) interactions with ruffling lamellipodia. More than 70% of the obstacle movements were caused by filopodial contractions in which the obstacle attached at the extreme distal end of a filopodium and moved only as the filopodium changed its extension. Filopodial contractions were characterized by frequent changes of obstacle velocity and direction. Contraction of a single filopodium is estimated to exert 50-90 microdyn of force, which can account for the pull exerted by chick sensory growth cones. Importantly, all five cases of growth cones growing over the top of obstacle neurites (i.e., geometry that mimics the usual growth cone/substrate interaction), were of the filopodial contraction type. Some 25% of obstacle movements occurred by a smooth backward movement along the top surface of growth cones. Both the appearance and rate of movements were similar to that reported for retrograde flow of cortical actin near the dorsal growth cone surface. Although these retrograde flow movements also exerted enough force to account for growth cone pulling, we did not observe such movements on ventral growth cone surfaces. Occasionally obstacles were moved by interaction with ruffling lamellipodia. However, we obtained no evidence for attachment of the obstacles to ruffling lamellipodia or for directed obstacle movements by this mechanism. These data suggest that chick sensory growth cones move forward by contractile activity of filopodia, i.e., isometric contraction on a rigid substrate. Our data argue against retrograde flow of actin producing traction force.

Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro.

HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.

Polarity orientation of axonal microtubules.

The polarity orientation of cellular microtubules is widely regarded to be important in understanding the control of microtubule assembly and microtubule-based motility in vivo. We have used a modification of the method of Heidemann and McIntosh (Nature (Lond.). 286:517-519) to determine the polarity orientation of axonal microtubules in postganglionic sympathetic fibers of the cat. In fibers from three cats we were able to visualize the polarity of 68% of the axonal microtubules; of these, 96% showed the same polarity orientation. Our interpretation is that the rapidly growing end of all axonal microtubules is distal to the cell body. We support Kirschner's hypothesis on microtubule organizing centers. (J. Cell Biol. 86:330-334), although this interpretation raises questions about the continuity of axonal microtubules. Our results are inconsistent with a number of models for axonal transport based on force production on the surface of microtubules in which the direction of force is determined by the polarity of microtubules.

Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.

Direct observations of the mechanical behaviors of the cytoskeleton in living fibroblasts.

Cytoskeletal proteins tagged with green fluorescent protein were used to directly visualize the mechanical role of the cytoskeleton in determining cell shape. Rat embryo (REF 52) fibroblasts were deformed using glass needles either uncoated for purely physical manipulations, or coated with laminin to induce attachment to the cell surface. Cells responded to uncoated probes in accordance with a three-layer model in which a highly elastic nucleus is surrounded by cytoplasmic microtubules that behave as a jelly-like viscoelastic fluid. The third, outermost cortical layer is an elastic shell under sustained tension. Adhesive, laminin-coated needles caused focal recruitment of actin filaments to the contacted surface region and increased the cortical layer stiffness. This direct visualization of actin recruitment confirms a widely postulated model for mechanical connections between extracellular matrix proteins and the actin cytoskeleton. Cells tethered to laminin-treated needles strongly resisted elongation by actively contracting. Whether using uncoated probes to apply simple deformations or laminin-coated probes to induce surface-to-cytoskeleton interaction we observed that experimentally applied forces produced exclusively local responses by both the actin and microtubule cytoskeleton. This local accomodation and dissipation of force is inconsistent with the proposal that cellular tensegrity determines cell shape.

A cytomechanical investigation of neurite growth on different culture surfaces.

We have examined the relationship between tension, an intrinsic stimulator of axonal elongation, and the culture substrate, an extrinsic regulator of axonal elongation. Chick sensory neurons were cultured on three substrata: (a) plain tissue culture plastic; (b) plastic treated with collagen type IV; and (c) plastic treated with laminin. Calibrated glass needles were used to increase the tension loads on growing neurites. We found that growth cones on all substrata failed to detach when subjected to two to threefold and in some cases 5-10-fold greater tensions than their self-imposed rest tension. We conclude that adhesion to the substrate does not limit the tension exerted by growth cones. These data argue against a "tug-of-war" model for substrate-mediated guidance of growth cones. Neurite elongation was experimentally induced by towing neurites with a force-calibrated glass needle. On all substrata, towed elongation rate was proportional to applied tension above a threshold tension. The proportionality between elongation rate and tension can be regarded as the growth sensitivity of the neurite to tension, i.e., its growth rate per unit tension. On this basis, towed growth on all substrata can be described by the simple linear equation: elongation rate = sensitivity x (applied tension - tension threshold) The numerical values of tension thresholds and neurite sensitivities varied widely among different neurites. On all substrata, thresholds varied from near zero to greater than 200 mudynes, with some tendency for thresholds to cluster between 100 and 150 mudynes. Similarly, the tension sensitivity of neurites varied between 0.5 and 5.0 microns/h/mudyne. The lack of significant differences among sensitivity or threshold values on the various substrata suggest to use that the substratum does not affect the internal "set points" of the neurite for its response to tension. The growth cone of chick sensory neurons is known to pull on its neurite. The simplest cytomechanical model would assume that both growth cone-mediated elongation and towed growth are identical as far as tension input and elongation rate are concerned. We used the equation above and mean values for thresholds and sensitivity from towing experiments to predict the mean growth cone-mediated elongation rate based on mean rest tensions. These predictions are consistent with the observed mean values.

Tension and compression in the cytoskeleton of PC 12 neurites.

We report in this article that the retraction of PC 12 neurites, unlike that of other cultured neurons, is due to tension within the neurite. Retraction is rapid and independent of metabolic energy. Transection of one arm of a branched neurite immediately causes the remaining arm to take up a new equilibrium position between attachment points. Similarly, detachment of one growth cone of a cell causes the cell body to move to a new equilibrium position between the remaining neurites. These observations provide direct evidence for the suspension of the cell soma among a network of tensioned neurites. We used retraction as an assay for neurite tension to examine the role of actin filaments and microtubules in neurite support and elongation. Our data suggest that microtubules (MTs) within PC 12 neurites are under compression, supporting tension within the actin network. Treatment of cells with drugs that disrupt actin networks, cytochalasin D or erythro-9-[3-(2-hydroxynonyl)]adenosine eliminates retraction regardless of the absence of MTs, lack of adhesion to the substratum, or integrity of the neurite. Conversely, stimulation of actin polymerization by injection of phalloidin causes retraction of neurites. Treatments that depolymerize MTs, nocodazole or cold, cause retraction of neurites, which suggests that microtubules support this tension, i.e., are under compression. Stabilization of MTs with taxol stabilizes neurites to retraction and under appropriate circumstances can drive neurite extension. Taxol-stimulated neurite extension is augmented by combined treatment with anti-actin drugs. This is consistent with the actin network's normally exerting a force opposite that of MT assembly. Cytochalasin and erythro-9-[3-(2-hydroxynonyl)] adenosine were found to increase slightly the dose of nocodazole required for MT depolymerization. This is consistent with the postulated balance of forces and also suggests that alteration of the compression borne by the microtubules could serve as a local regulator for MT polymerization during neurite outgrowth.

Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells.

The outgrowth of neurites from rat PC12 cells stimulated by combined treatment of nerve growth factor (NGF) with cAMP is significantly more rapid and extensive than the outgrowth induced by either factor alone. We have compared the responses of PC12 cells under three different growth conditions, NGF alone, cAMP alone, and combined treatment, with respect to surface morphology, rapidity of neurite outgrowth, and stability of neurite microtubules, to understand the synergistic action of NGF and cAMP on PC12. Surface events at early times in these growth conditions varied, suggesting divergent pathways of action of NGF and cAMP. This suggestion is strongly supported by the finding that cells exposed to saturating levels of dibutyryl cAMP without substantial neurite outgrowth initiated neurites within 5 min of NGF. This response has been adopted as a convenient assay for NGF. Neurites that regenerated in the three growth conditions showed marked differences in stability to treatments that depolymerize microtubules. The results indicate that microtubules in cells treated with both NGF and cAMP are significantly more stable than in either growth factor alone. We suggest that a shift of the assembly equilibrium favoring tubulin assembly is a necessary prerequisite for the initiation of neurites by PC12.

The cytomechanics of axonal elongation and retraction.

Neurites of PC12 and chick dorsal root ganglion neurons behave as viscoelastic solids in response to applied forces. This passive behavior can be modeled with three mechanical elements; a relatively stiff, undamped spring in series with a Voight element composed of a less stiff spring in parallel with a dashpot. In response to applied tensions greater than 100 microdynes, PC12 cells show lengthening behavior distinct from and in addition to the passive viscoelastic response. We interpret this as "towed growth" (Bray, D. 1984. Dev. Biol. 102:379-389) because the neurites can become twice as long without obvious thinning of the neurite and because in two cases neurite tensions fell below original rest tensions, a result that cannot be obtained with passive viscoelastic elements. The rate of towed growth showed a linear dependence of growth rate with applied tensions in 8 of 12 PC12 neurites exposed to applied tension greater than 100 microdynes. Both PC12 and chick sensory neurons showed evidence of retraction when neurite tensions were suddenly diminished. This response was measured as tension recovery after slackening in chick sensory neurites. In 62% of the cases, tension recovery exceeded and sometimes doubled the preexperimental steady-state tension. Our data indicate that this response is active tension generation by the neurite shaft. We conclude that neurite length is regulated by axial tension in both elongation and retraction. Our data suggest a three-way controller: above some tension set point, the neurite is stimulated to elongate. Below some different, lower tension threshold the neurite is stimulated to retract. Between these two tension thresholds, the neurite responds passively as a viscoelastic solid.

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules.

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.

Tension and compression in the cytoskeleton of PC-12 neurites. II: Quantitative measurements.

We assessed the mechanical properties of PC-12 neurites by applying a force with calibrated glass needles and measured resulting changes in neurite length and deflection of the needle. We observed a linear relationship between force and length change that was not affected by multiple distensions and were thus able to determine neurite spring constants and initial, nondistended, rest tensions. 81 out of 82 neurites showed positive rest tensions ranging over three orders of magnitude with most values clustering around 30-40 mu dynes. Treatment with cytochalasin D significantly reduced neurite rest tensions to an average compression equal to 14% of the former tension and spring constants to an average of 17% of resting values. Treatment with nocodazole increased neurite rest tensions to an average of 282% of resting values but produced no change in spring constant. These observations suggest a particular type of complementary force interaction underlying axonal shape; the neurite actin network under tension and neurite microtubules under compression. Thermodynamics suggests that microtubule (MT) assembly may be regulated by changes in compressive load. We tested this effect by releasing neurite attachment to a polylysine-coated surface with polyaspartate, thus shifting external compressive support onto internal elements, and measuring the relative change in MT polymerization using quantitative Western blotting. Neurons grown on polylysine or collagen without further treatment had a 1:2 ratio of soluble to polymerized tubulin. When neurites grown on polylysine were treated with 1% polyaspartate for 15-30 min, 80% of neurites retracted, shifting the soluble: polymerized tubulin ratio to 1:1. Polyaspartate treatment of cells grown on collagen, or grown on polylysine but treated with cytochalasin to reduce tension, caused neither retraction nor a change in the soluble:polymerized tubulin ratio. We suggest that the release of adhesion to the dish shifted the compressive load formerly borne by the dish onto Mts causing their partial depolymerization. Our observations are consistent with the possibility that alterations in MT compression during growth cone advance integrates MT assembly with the advance.

Spatial organization of axonal microtubules.

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.

F-actin and microtubule suspensions as indeterminate fluids.

The viscosity of F-actin and microtubule suspensions has been measured as a function of shear rate with a Weissenberg rheogoniometer. At shear rates of less than 1.0 per second the viscosity of suspensions of these two structural proteins is inversely proportional to shear rate. These results are consistent with previous in vivo measurements of the viscosity of cytoplasm. This power law implies that shear stress is independent of shear rate; that is, shear stress is a constant at all shear rates less than 1.0 per second. Thus the flow profile of these fluids is indeterminate, or nearly so. This flow property may explain several aspects of intracellular motility in living cells. Possible explanations for this flow property are based on a recent model for semidilute suspensions of rigid rods or a classical friction model for liquid crystals.

Cardiac atria of BIO 14.6 hamsters are deficient in natriuretic factor.

The hearts of 220-day-old hamsters of the BIO 14.6 strain are deficient in atrial natriuretic factor; saline extracts of atria produce one-third the natriuretic and diuretic effects of extracts of atria from age-matched normal hamsters. BIO 14.6 hamsters are known to develop congestive heart failure with edema when they are about 200 days old, and the venous congestion and edema are preventable by parabiosis with normal hamsters. The humoral mediator, the deficiency of which causes venous congestion and edema in BIO 14.6 hamsters, may be atrial natriuretic factor.

Growth cone motility.

The exact nature of growth cone motility is far from understood but progress has been made in several areas. It now appears that growth cones pull and not push; we will review the biophysical basis of growth cone movement. Current ideas on the regulation of growth cone motility and the relationship between motility and axon pathfinding are also discussed.