Kinesin associates with anterogradely transported membranous organelles in vivo

Biochemical, pharmacological and immunocytochemical studies have implicated the microtubule-activated ATPase, kinesin, in the movement of membrane bounded organelles in fast axonal transport. In vitro studies suggested that kinesin moves organelles preferentially in the anterograde direction, but data about the function and precise localization of kinesin in the living axon were lacking. The current study was undertaken to establish whether kinesin associates with anterograde or retrograde moving organelles in vivo. Peripheral nerves were ligated to produce accumulations of organelles moving in defined directions. Regions proximal (anterograde) and distal (retrograde) to the ligation were analyzed for kinesin localization by immunofluorescence, and by immunogold electron microscopy using ultracryomicrotomy. Substantial amounts of kinesin were associated with anterograde moving organelles on the proximal side, while significantly less kinesin was detected distally. Statistical analyses indicated that kinesin was mostly associated with membrane-bounded organelles. These observations indicate that axonal kinesin is primarily associated with anterograde moving organelles in vivo.     

The movement of membranous organelles in axons. Electron microscopic identification of anterogradely and retrogradely transported organelles

To identify the structures to be rapidly transported through the axons, we developed a new method to permit local cooling of mouse saphenous nerves in situ without exposing them. By this method, both anterograde and retrograde transport were successfully interrupted, while the structural integrity of the nerves was well preserved. Using radioactive tracers, anterogradely transported proteins were shown to accumulate just proximal to the cooled site, and retrogradely transported proteins just distal to the cooled site. Where the anterogradely transported proteins accumulated, the vesiculotubular membranous structures increased in amount inside both myelinated and unmyelinated axons. Such accumulated membranous structures showed a relatively uniform diameter of 50--80 nm, and some of them seemed to be continuous with the axonal smooth endoplasmic reticulum (SER). Thick sections of nerves selectively stained for the axonal membranous structures revealed that the network of the axonal SER was also packed inside axons proximal to the cooled site. In contrast, large membranous bodies of varying sizes accumulated inside axons just distal to the cooled site, where the retrogradely transported proteins accumulated. These bodies were composed mainly of multivesicular bodies and lamellated membranous structures. When horseradish peroxidase was administered in the distal end of the nerve, membranous bodies showing this activity accumulated, together with unstained membranous bodies. Hence, we are led to propose that, besides mitochondria, the membranous components in the axon can be classified into two systems from the viewpoint of axonal transport: "axonal SER and vesiculotubular structures" in the anterograde direction and "large membranous bodies" in the retrograde direction.     

Differential phosphorylation in vivo of cytoplasmic dynein associated with anterogradely moving organelles

Two microtubule-stimulated ATPases, cytoplasmic dynein, and kinesin, are believed to be responsible for the intracellular movement of membrane-bound organelles in opposite directions along microtubules. An unresolved component of this model is the mechanism by which cells regulate these two motors to direct various membrane-bound organelles to their proper locations. To determine if phosphorylation may play a role in the regulation of cytoplasmic dynein, the in vivo phosphorylation state of cytoplasmic dynein from two cellular pools was examined. The entire cellular pool of brain cytoplasmic dynein was metabolically labeled by the infusion of [32P]orthophosphate into the cerebrospinal fluid of rat brain ventricles. To characterize the phosphorylation of dynein associated with anterograde membrane-bound organelles, the optic nerve fast axonal transport system was used. Using a monoclonal antibody to the 74-kD polypeptide of brain cytoplasmic dynein, the native dynein complex was immunoprecipitated from the radiolabled tissue extracts. Autoradiographs of one and two dimensional gels showed labeling of nearly all of the polypeptide isoforms of cytoplasmic dynein from rat brain. These polypeptides are phosphorylated on serine residues. Comparison of the amount of 32P incorporated into the dynein polypeptides revealed differences in the phosphorylation of dynein polypeptides from the anterograde and the cellular pools. Most interestingly, the 530-kD heavy chain of dynein appears to be phosphorylated to a lesser extent in the anterograde pool than in the cellular pool. Since the anterograde pool contains inactive dynein, while the entire cellular pool contains both inactive and active dynein, these results are consistent with the hypothesis that phosphorylation regulates the functional activity of cytoplasmic dynein.     

Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C)

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.     

Age-Dependent Organotypic Expression of Microtubule-Associated Proteins (MAP1, MAP2, and MAP5) in Rat Brain

Age-dependent changes in the distribution of microtubule-associated proteins (MAPs) were analyzed in young (3-months, N = 3) and old (24-months, N = 3) rat brain. In the young rats, MAP1 and MAP5 exhibited prominent immunostaining in the perikarya and dendrites whereas MAP2 was selectively localized in the dendrites. In the cerebellum, MAP2 was preferentially localized in finer and distal branches of Purkinje cell dendrites and in punctate deposits surrounding glomeruli. In general, aging resulted in obvious declines in MAP2- >> MAP1- and MAP5-immunoreactivities in the hippocampus and parietal cortex but no change in cerebellum. The results indicate that: (1) hippocampus is the most affected and cerebellum is the least affected region with regard to declines in MAPs-immunoreactivities in the aged rat brain; (2) dendrite-specific MAP2 is almost completely depleted from most dendrites in the hippocampus and cortex. In summary, loss of MAP2-immunoreactivity in the affected brain areas may be associated with age-related impairment of synaptic plasticity, cognition and memory functions.     

Selective MAP1LC3C (LC3C) autophagy requires noncanonical regulators and the C-terminal peptide

LC3s are canonical proteins necessary for the formation of autophagosomes. We have previously established that two paralogs, LC3B and LC3C, have opposite activities in renal cancer, with LC3B playing an oncogenic role and LC3C a tumor-suppressing role. LC3C is an evolutionary late gene present only in higher primates and humans. Its most distinct feature is a C-terminal 20-amino acid peptide cleaved in the process of glycine 126 lipidation. Here, we investigated mechanisms of LC3C-selective autophagy. LC3C autophagy requires noncanonical upstream regulatory complexes that include ULK3, UVRAG, RUBCN, PIK3C2A, and a member of ESCRT, TSG101. We established that postdivision midbody rings (PDMBs) implicated in cancer stem-cell regulation are direct targets of LC3C autophagy. LC3C C-terminal peptide is necessary and sufficient to mediate LC3C-dependent selective degradation of PDMBs. This work establishes a new noncanonical human-specific selective autophagic program relevant to cancer stem cells.     

Assembly and architecture of biogenesis of lysosome-related organelles complex-1 (BLOC-1)

BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.     

Orexin-B-like immunoreactivity localizes in both luteinizing-hormone-containing cells and melanin-concentrating hormone-containing fibers in the red-bellied piranha (Pygocentrus nattereri) pituitary

We examined orexin-like immunoreactivity in the pituitary of the red-bellied piranha (Pygocentrus nattereri). Orexin-B-immunoreactive (IR) cells corresponded to luteinizing hormone (LH)-containing cells in the pars distalis, and orexin-B-IR fibers corresponded to melanin-concentrating hormone (MCH)-containing fibers in the pars nervosa. In the pars distalis, orexin-B-IR puncta that were also immunoreactive for MCH were observed around the orexin-B-IR cells. In the ventral hypothalamus, orexin-B-IR and MCH-IR neurons were found in the nucleus lateralis tuberis. Immunoelectron-microscopic analysis revealed that the orexin-B-like substance co-localized with LH in secretory granules and with MCH in MCH-containing neurons. Some of the MCH secreted in the pituitary might participate in the modulation of LH secretion from the gonadotrophs, together with orexin-B, leading to food intake by the stimulation of growth hormone secretion from the somatotrophs.     

Helicobacter pylori heat shock protein B (HspB) localizes in vivo in the gastric mucosa and MALT lymphoma

Heat shock protein B (HspB) is one of the dominant proteins recognized by most Helicobacter pylori-infected persons and is being considered as potential candidates for subunit vaccines. In the present study we describe the generation of an antibody against HspB and its use in immunohistochemical assays on gastric biopsies. We have demonstrated that our rabbit polyclonal antibody against HspB did not recognize any protein in lysates from a lung human epithelial cell (H1299) line and did not cross-react with the other members of human heat shock proteins. Secondly, we have observed that in gastric biopsies, HspB immunostaining was present inside the cytoplasm of human epithelial cells with a particular localization in the apical portion of gastric epithelial cells other than in the extracellular spaces among gastric cells of human stomach. Finally, we have demonstrated a cytoplasmic HspB immunostaining in groups of neoplastic cells of MALT lymphoma. In conclusion, our observations suggest a possible involvement of HspB in the pathogenesis of H. pylori-related pathologies such as gastritis, ulcer and gastric cancer.     

Insulin regulates MAP kinase phosphatase-1 induction in Hirc B cells via activation of both extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK)

Previously, we have reported that insulin induces the expression of the dual-specificity tyrosine phosphatase Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) and that this may represent a negative feedback mechanism to regulate insulin-stimulated MAP kinase activity. In this work, the mechanism of regulation of MKP-1 expression by insulin was examined, particularly the role of the MAP kinase superfamily. Inhibition of the ERK pathway attenuated insulin-stimulated MKP-1 mRNA expression. Expression of dominant negative molecules of the JNK pathway also abolished insulin-stimulated MKP-1 expression. However, inhibition of p38^MAPK activity by SB202190 had no effect on insulin-stimulated MKP-1 induction. Simultaneous inhibition of the ERK and JNK pathways abolished the ability of insulin to stimulate MKP-1 expression, however, this combined inhibition was neither additive nor synergistic, suggesting these pathways converge to act on a common final effector. In conclusion, induction of MKP-1 mRNA expression in Hirc B cells by insulin requires activation of both the ERK and JNK pathways, but not p38^MAPK.     

Inhibition of MAP kinase kinase (MEK) results in an anti-inflammatory response in vivo

The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.     

Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2.     

Functional Impairment in Protein Kinase C by RACK1 (Receptor for Activated C Kinase 1) Deficiency in Aged Rat Brain Cortex

Abstract: Several laboratories have reported a lack of protein kinase C (PKC) activation in response to various stimuli in the brain of aged rats. It has been suggested that changes in lipid membrane composition could be related to this functional deficit. However, recent evidence has indicated that the translocation of PKC to the different subcellular compartments is controlled by protein-protein interactions. Recently, a class of proteins, termed receptors for activated C kinase (RACKs), have been described that bind PKC. The present study was conducted to determine whether alterations in RACK1, the best-characterized member of RACKs, were associated with changes in translocation and expression of PKC. Quantitative immunoblotting revealed that RACK1 content was decreased by ∼50% in aged rat brain cortex, compared with that in adult and middle-aged animals. The levels of calcium-independent PKCδ and ε, interacting with RACK1, and related calcium-independent PKC activity were not modified by the aging process. By comparison, phorbol ester-stimulated translocation of this activity and of PKCδ and ε immunoreactivity was absent in cortex from aged animals, as well as the translocation of the calcium-dependent PKCβ, also known to interact with RACK1. These results indicate that a deficit in RACK1 may contribute to the functional impairment in PKC activation observed in aged rat brain.     

A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro

A gene encoding a putative guanosine 3′,5′-bispyrophosphate (ppGpp) synthase–degradase, designated Cr-RSH, was identified in the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii. The encoded Cr-RSH protein possesses a putative chloroplast-targeting signal at its NH₂-terminus, and translocation of Cr-RSH into chloroplasts isolated from C.reinhardtii was demonstrated in vitro. The predicted mature region of Cr-RSH exhibits marked similarity to eubacterial members of the RelA–SpoT family of proteins. Expression of an NH₂-terminal portion of Cr-RSH containing the putative ppGpp synthase domain in a relA, spoT double mutant of Escherichia coli complemented the growth deficits of the mutant cells. Chromatographic analysis of ³²P-labeled cellular mononucleotides also revealed that expression of Cr-RSH in the mutant bacterial cells resulted in the synthesis of ppGpp. SpoT, which catalyzes (p)ppGpp degradation, is dispensable in E.coli only if cells also lack RelA, which possesses (p)ppGpp synthase activity. The complementation analysis thus indicated that Cr-RSH possesses both ppGpp synthase and degradase activities. These results represent the first demonstration of ppGpp synthase–degradase activities in a eukaryotic organism, and they suggest that eubacterial stringent control mediated by ppGpp has been conserved during evolution of the chloroplast from a photosynthetic bacterial symbiont.     

Measurement of antibody-dependent binding, proteolysis, and turnover of C1s on liposomal antigens localizes the fluidity-dependent step in C1 activation

The antibody-dependent binding and activation of the first component of human complement (C1) by liposomes containing nitroxide spin-label lipid haptens have been simultaneously measured. The liposomes were either fluid (dimyristoylphosphatidylcholine) or solid (dipalmitoylphosphatidylcholine) at the temperature of the experiments (32 degrees C). In 10 minutes fluid liposomes activate 40% of the C1 whereas solid liposomes only activate 10% of the C1. The fraction of C1 bound at the end of the activation incubation is approx. 2% for fluid liposomes and approx. 4% for solid liposomes. This binding is consistent with the relative amounts of antibody which bind to these two types of liposomes. These results demonstrate turnover of C1 or C1r2s2 on the liposome surface. It is concluded that the differential activation of C1 is due to a difference in the rate of activation of C1 after it is bound to the liposome surface. Lower limits for the activation rate constant for C1 bound to fluid and solid liposomes are estimated to be 8 X 10(-2) s-1 and 1 X 10(-2) s-1, respectively.