Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.     

Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis

Synthesis of new proteins is required to regenerate full length Chlamydomonas flagella after deflagellation. Using gametes, which have a low basal level of protein synthesis, it has been possible to label and detect the synthesis of many flagellar proteins in whole cells. The deflagellation-induced synthesis of the tubulins, dyneins, the flagellar membrane protein, and at least 20 other proteins which co- migrate with proteins in isolated axonemes, can be detected in gamete cytoplasm, and the times of initiation and termination of synthesis for each of the proteins can be studied. The nature of the signal that stimulates the cell to initiate flagellar protein synthesis is unknown. Flagellar regeneration and accompanying pool depletion are not necessary for either the onset or termination of flagellar protein synthesis, because colchicine, which blocks flagellar regeneration, does not change the pattern of proteins synthesized in the cytoplasm after deflagellation or the timing of their synthesis. Moreover, flagellar protein synthesis is stimulated after cells are chemically induced to resorb their flagella, indicating that the act of deflagellation itself is not necessary to stimulate synthesis. Methods were defined for inducing the cells to resorb their flagella by removing Ca++ from the medium and raising the concentration of K+ or Na+. The resorption was reversible and the flagellar components that were resorbed could be re-utilized to assemble flagella in the absence of protein synthesis. This new technique is used in this report to study the control of synthesis and assembly of flagella.     

Formation and maintenance of tubular membrane projections require mechanical force, but their elongation and shortening do not require additional force

Living cells develop their own characteristic shapes depending on their physiological functions, and their morphologies are based on the mechanical characteristics of the cytoskeleton and of membranes. To investigate the role of lipid membranes in morphogenesis, we constructed a simple system that can manipulate liposomes and measure the forces required to transform their shapes. Two polystyrene beads (1 microm in diameter) were encapsulated in giant liposomes and were manipulated using double-beam laser tweezers. Without any specific interaction between the lipid membrane and beads, mechanical forces could be applied to the liposome membrane from the inside. Spherical liposomes transformed into a lemon shape with increasing tension, and tubular membrane projections were subsequently generated in the tips at either end. This process is similar to the liposomal transformation caused by elongation of encapsulated cytoskeletons. In the elongation stage of lemon-shaped liposomes, the force required for the transformation became larger as the end-to-end length increased. Just before the tubular membrane was generated, the force reached the maximum strength (approximately 11 pN). However, immediately after the tubular membrane developed, the force suddenly decreased and was maintained at a constant strength (approximately 4 pN) that was independent of further tube elongation or shortening, even though there was no excess membrane reservoir as occurs in living cells. When the tube length was shortened to approximately 2 microm, the liposome reversed to a lemon shape and the force temporarily increased (to approximately 7 pN). These results indicate that the simple application of mechanical force is sufficient to form a protrusion in a membrane, that a critical force and length is needed to form and to maintain the protrusion, and suggest that the lipid bilayer itself has the ability to buffer the membrane tension.     

Protein that binds to the distal,but not to the proximal,CCAAT of the human thymidine kinase gene promoter

Mobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK-CBP) was also present in two human cell lines. Significantly more DTK-CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK^?ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.     

Telomerase activation induces elongation of the telomeric single-stranded overhang, but does not prevent chromosome aberrations in human vascular endothelial cells

Chromosome aberrations such as loss of chromosome 13 were frequently observed in human endothelial cells from umbilical cord veins (HUVEC). A recent study showed that the length of telomeric single-stranded 3'-overhangs (G-tails) is more important as an essential structure for chromosome maintenance than the net telomere length in telomere t-loop formation. Here, we have examined G-tail length using G-tail telomere HPA in normal and hTERT-transduced HUVECs. We found that forced expression of hTERT in HUVEC induced G-tail as well as total telomere length elongation. G-tail length was well correlated with total telomere length. However, hTERT introduction did not prevent chromosome aberrations such as loss of chromosome 13. Normal characteristics such as morphology, up-regulation of vWF, and tube formation were observed in hTERT-HUVEC as in young normal HUVEC. These results show that chromosome aberrations in HUVEC are independent of telomere G-tail and total telomere attrition.     

Release of peptide YY by fat in the proximal but not distal gut depends on an atropine-sensitive cholinergic pathway

Peptide YY (PYY) is released when PYY cells in short term culture are exposed to fat suggesting that this peptide may be released by fat in the distal gut without neural stimulation. PYY is also released by fat in the proximal 1/2 of small intestine. To test the hypothesis that the release of PYY by fat in the proximal but not distal intestine may depend on an atropine-sensitive, cholinergic pathway, PYY levels were compared in four dogs equipped with duodenal and mid-intestinal fistulas when 60 mM oleate was perfused into either the proximal (between fistulas) or distal (beyond mid-intestinal fistula) 1/2 of gut at 2 ml/min for 120 min with intravenous administration of saline or atropine. We found that, when fat was confined to the proximal 1/2 of the intestine, PYY release was reduced following intravenous atropine when compared with saline (p<0.01). However, when fat was confined to the distal 1/2 of the intestine, PYY release was not affected by the intravenous atropine. We conclude that PYY release by fat in the proximal but not distal intestine depends on an atropine-sensitive, cholinergic pathway.     

Ultrastructure of the choroid plexus epithelium of pigeons treated with drugs: II. Effect of cytochalasin D and colchicine

A remarkable projection of bleblike protrusions, the expulsion of organelles into the protrusions formed on the apical surface, and the separation into the ventricular lumen of these protrusions was the general cellular response of choroidal epithelial cells to intravenous injection of cytochalasin D (CD). The compact microfilament mass and agglomeration of microtubules at the base of the cluster of protrusions reflect the results of cell contraction and displacement of microfilaments induced by CD. In earlier stages after intravenous injections of colchicine, an obvious increase in the number of various-sized vesicles, vacuoles, and lysosomes in the Golgi region was detected. In the later stages, these organelles were seen to accumulate in the basal portion of the epithelial cells. These changes were accompanied by an increase in vacuoles and the disorganization and displacement of the Golgi complex, and they coincided with a decrease in the number of microtubules in apical and basal cytoplasm. These findings suggest that the action of colchicine results in destruction of the three-dimensional architecture between cytoskeletal network and cell organelles. The present results suggest that the cytoskeletal network plays a role in the spatial coordination of the three-dimensional architecture of cell organelles. The study also indicates that the structural differences in the ventricles of the choroid plexus in drug-treated pigeons are manifestations of regional functional specialization in different parts of the ventricular system.     

An essential role for the proximal but not the distal cytoplasmic tail of glycoprotein M in murid herpesvirus 4 infection

Murid herpesvirus-4 (MuHV-4) provides a tractable model with which to define common, conserved features of gamma-herpesvirus biology. The multi-membrane spanning glycoprotein M (gM) is one of only 4 glycoproteins that are essential for MuHV-4 lytic replication. gM binds to gN and is thought to function mainly secondary envelopment and virion egress, for which several predicted trafficking motifs in its C-terminal cytoplasmic tail could be important. We tested the contribution of the gM cytoplasmic tail to MuHV-4 lytic replication by making recombinant viruses with varying C-terminal deletions. Removing an acidic cluster and a distal YXXPhi motif altered the capsid distribution somewhat in infected cells but had little effect on virus replication, either in vitro or in vivo. In contrast, removing a proximal YXXPhi motif as well completely prevented productive replication. gM was still expressed, but unlike its longer forms showed only limited colocalization with co-transfected gN, and in the context of whole virus appeared to support gN expression less well. We conclude that some elements of the gM cytoplasmic tail are dispensible for MuHV-4 replication, but the tail as a whole is not.     

Inflection points of cardiovascular responses and oxygenation are correlated in the distal but not the proximal portions of muscle during incremental exercise.

To test whether there is a regional difference in the exercise pressor reflex within a given muscle, we investigated the relationship between the inflection points of cardiovascular responses and muscle oxygenation during exercise. Seven subjects performed incremental exercise, which consisted of incremental 30-s static knee extensions, each separated by 30 s of recovery. The workload started at 5% maximal voluntary contraction (MVC) and increased by 5% MVC for each increment until exhaustion. Changes (Delta) in the concentrations (denoted by brackets) of oxygenated Hb (O2Hb) and deoxygenated Hb (HHb) were monitored in proximal and distal portions of the vastus lateralis by near-infrared spectroscopy. The inflection points of mean arterial pressure (MAP), calf vascular resistance (CVR), and muscle deoxygenation index (Delta[O2Hb-HHb]) were calculated as the intersection point of two regression equations obtained at lower and higher workloads. The inflection point of Delta[O2Hb-HHb] differed significantly between proximal and distal portions (28.5 +/- 3.0 vs. 39.5 +/- 3.0%MVC, P < 0.05). Linear regression analysis showed significant correlations between the inflection point of Delta[O2Hb-HHb] in the distal portion and MAP (r = 0.89; P < 0.01) and CVR (r = 0.89; P < 0.05), but no significant relationship between the inflection point in the proximal portion and MAP or CVR. These data show that the inflection point of muscle deoxygenation differs between proximal and distal portions within the vastus lateralis during incremental exercise and suggest that the distal portion of the vastus lateralis contributes more to the pressor response than does the proximal portion.     

On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force

As anaphase began, mitotic PtK1 and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (approximately 16 degrees C) or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtubules (MTs) were relatively stable after permeabilization and MT assembly was usually promoted in asters. Astral and kinetochore MTs were sensitive to MT disassembly conditions, and shortening of kinetochore MTs always accompanied reactivation of anaphase A. Interphase and interzonal spindle MTs were relatively stable to cold and calcium until extraction of cells was promoted by longer periods in the PM, or by higher concentrations of detergent. Since we cannot envisage how both cold treatment or relatively high calcium levels can reactivate spindle motility in quiescent, permeabilized, and presumably energy-depleted cells, we conclude that anaphase A is powered by energy stored in the spindle. The nucleotide triphosphates effective in reactivating anaphase A could be necessary for the kinetochore MT disassembly without which anaphase movement cannot proceed.     

Modulation of interleukin 1 production by activated macrophages: in vitro action of hydrocortisone, colchicine, and cytochalasin B

In vitro cultured irradiated rat peritoneal macrophages (XPCs) secreted markedly more Interleukin 1 (IL-1) when pretreated with carrageenan. Exposure of cultures of carrageenanpretreated as well as untreated XPCs to hydrocortisone (OHC) produced a dose-dependent inhibition of IL-1 secretion. OHC at concentrations from 10^?4 to 10^?6M completely blocked the enhanced IL-1 secretion induced by carrageenan pretreatment. These observations suggest that the complex immunosuppressive effects of glucocorticoids could partly result from their capability to control IL-1 production. Colchicine, a microtubule depolymerizing agent, markedly enhanced IL-1 secretion in cultures of both carrageenan-pretreated and untreated XPCs. Stimulatory effects were dose dependent and maximum stimulation occurred at 10^?4M, i.e., at a concentration known to profoundly affect the microtubular function. Lumicolchicine, however, was inactive. Cytochalasin B, a microfilament disrupting agent and inhibitor of phagocytosis, elicited a dose-dependent increase of IL-1 secretion in cultures of carrageenanactivated XPCs but was inhibitory for unstimulated XPCs. Although mechanisms by which Colchicine and Cytochalasin B act in stimulating IL-1 production remain unclear, our study demonstrates that production and secretion of IL-1 do not require participation of intact components of the cytoskeleton.     

Effect of the microtubule disrupting agents,colchicine and vinblastine,on seminiferous tubule structure in the rat

Injections of colchicine or vinblastine were given intratesticularly and rats sacrificed 6 and 12 hr later. Colchicine and vinblastine produced identical morphological patterns of response in the seminiferous tubules resulting in arrest of germcell mitoses and meioses and a rapid depletion of the microtubules normally found within the Sertoli cell. Sloughing of cells into the lumen of seminiferous tubules was the most prominent feature noted. Germ cells and portions of the apical Sertoli cells were frequently sloughed together where they remained in close association. Usually germ cells and associated Sertoli cell fragments were cleaved from the wall of the seminiferous tubule at a level between dissimilar generations of germ cells, e.g. between spermatocytes and spermatids. This selective sloughing probably occurred as the result of the support normally provided by intercellular bridges which link clones of like germ cell types. Sequential steps in the process leading to sloughing of Sertoli-germ cell associations could be inferred from observations made in plastic 1 μm sections. Cell sloughing at 12 hr post-injection was generally more extensive. It was frequently noted that germ cells and the apical portions of Sertoli cells had been extruded to the level of the most adluminal tight junctions forming the blood-testis barrier. It was concluded that disruption of Sertoli microtubules was responsible for sloughing of Sertoli fragments and associated germ cells, and that the cytoskeletal support of the Sertoli cell was, at least in part, dependent upon the integrity of Sertoli microtubules. The Sertoli cell could not round-up after loss of its cytoskeletal support, due to the numerous attachment devices known to link it with various apically positioned germ cells. Thus, the cell was severed at some point along its delicate apical processes, as the consequence of forces produced by the ‘rounding-up’ process. Long-term sacrifice after vinblastine or colchicine treatment allowed the Sertoli cells to regain microtubules and long processes but not their typical configuration. Spermatogenesis remained severely impaired.     

Mg‐dechelatase is involved in the formation of photosystem II but not in chlorophyll degradation in Chlamydomonas reinhardtii

The STAY‐GREEN (SGR) gene encodes Mg‐dechelatase which catalyzes the conversion of chlorophyll (Chl) a to pheophytin (Pheo) a. This reaction is the first and most important regulatory step in the Chl degradation pathway. Conversely, Pheo a is an indispensable molecule in photosystem (PS) II, suggesting the involvement of SGR in the formation of PSII. To investigate the physiological functions of SGR, we isolated Chlamydomonas sgr mutants by screening an insertion‐mutant library. The sgr mutants had reduced maximum quantum efficiency of PSII (F_v/F_m) and reduced Pheo a levels. These phenotypes were complemented by the introduction of the Chlamydomonas SGR gene. Blue Native polyacrylamide gel electrophoresis and immunoblotting analysis showed that although PSII levels were reduced in the sgr mutants, PSI and light‐harvesting Chl a/b complex levels were unaffected. Under nitrogen starvation conditions, Chl degradation proceeded in the sgr mutants as in the wild type, indicating that ChlamydomonasSGR is not required for Chl degradation and primarily contributes to the formation of PSII. In contrast, in the Arabidopsis sgr triple mutant (sgr1 sgr2 sgrL), which completely lacks SGR activity, PSII was synthesized normally. These results suggest that the Arabidopsis SGR participates in Chl degradation while the ChlamydomonasSGR participates in PSII formation despite having the same catalytic property.     

Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D

Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events. Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured. The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane. Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01). Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001). In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension. The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension. These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D. The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux.     

Ultraviolet irradiation induces the accumulation of chondroitin sulfate, but not other glycosaminoglycans, in human skin

Ultraviolet (UV) light alters cutaneous structure and function. Prior work has shown loss of dermal hyaluronan after UV-irradiation of human skin, yet UV exposure increases total glycosaminoglycan (GAG) content in mouse models. To more fully describe UV-induced alterations to cutaneous GAG content, we subjected human volunteers to intermediate-term (5 doses/week for 4 weeks) or single-dose UV exposure. Total dermal uronyl-containing GAGs increased substantially with each of these regimens. We found that UV exposure substantially increased dermal content of chondroitin sulfate (CS), but not hyaluronan, heparan sulfate, or dermatan sulfate. UV induced the accumulation of both the 4-sulfated (C4S) and 6-sulfated (C6S) isoforms of CS, but in distinct distributions. Next, we examined several CS proteoglycan core proteins and found a significant accumulation of dermal and endothelial serglycin, but not of decorin or versican, after UV exposure. To examine regulation in vitro, we found that UVB in combination with IL-1α, a cytokine upregulated by UV radiation, induced serglycin mRNA in cultured dermal fibroblasts, but did not induce the chondroitin sulfate synthases. Overall, our data indicate that intermediate-term and single-dose UVB exposure induces specific GAGs and proteoglycan core proteins in human skin in vivo. These molecules have important biologic functions and contribute to the cutaneous response to UV.     

Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.     

Light quality influences germination, root growth and hypocotyl elongation in somatic embryos but not in seedlings of norway spruce

Summary We studied how light from different light sources influences germination and postgerminative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst). Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 μmol m⁻² s⁻¹. After 6 wk the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 wk, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 wk, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.