A Class VI Unconventional Myosin Is Associated with a Homologue of a Microtubule-binding Protein,Cytoplasmic Linker Protein–170, in Neurons and at the Posterior Pole of Drosophila Embryos

Abstract. Coordination of cellular organization requires the interaction of the cytoskeletal filament systems. Recently, several lines of investigation have suggested that transport of cellular components along both microtubules and actin filaments is important for cellular organization and function. We report here on molecules that may mediate coordination between the actin and microtubule cytoskeletons. We have identified a 195-kD protein that coimmunoprecipitates with a class VI myosin, Drosophila 95F unconventional myosin. Cloning and sequencing of the gene encoding the 195-kD protein reveals that it is the first homologue identified of cytoplasmic linker protein (CLIP)–170, a protein that links endocytic vesicles to microtubules. We have named this protein D-CLIP-190 (the predicted molecular mass is 189 kD) based on its similarity to CLIP-170 and its ability to cosediment with microtubules. The similarity between D-CLIP-190 and CLIP-170 extends throughout the length of the proteins, and they have a number of predicted sequence and structural features in common. 95F myosin and D-CLIP-190 are coexpressed in a number of tissues during embryogenesis in Drosophila. In the axonal processes of neurons, they are colocalized in the same particulate structures, which resemble vesicles. They are also colocalized at the posterior pole of the early embryo, and this localization is dependent on the actin cytoskeleton. The association of a myosin and a homologue of a microtubule-binding protein in the nervous system and at the posterior pole, where both microtubule and actin-dependent processes are known to be important, leads us to speculate that these two proteins may functionally link the actin and microtubule cytoskeletons.Global organization of the cell and the coordination of its physiology requires interaction between different cytoskeletal systems. During interphase, a typical eukaryotic cell has microtubules emanating from the centrosome located near the nucleus, which extend to the periphery of the cell, presumably interacting with the cortical actin filament meshwork. Microtubules during interphase are thought to be mainly required for the organization of the membrane systems (e.g., vesicular traffic and organelle movement). The actin-rich cortex is important for maintaining cell shape and for cellular movement.There is increasing evidence of coordination between the actin and the microtubule cytoskeletons (Langford, 1995; Koonce, 1996). Data from a number of systems suggests that many cell types use a combination of microtubule and actin filament–based transport in vesicle and organelle trafficking. It is well established that microtubules are required for long distance transport of cellular components. In contrast, the actin cytoskeleton is thought to be required for more local traffic. The best evidence for transport along both cytoskeletal systems is in neurons. Vesicles appear to be transported along actin filaments in mammalian growth cones (Evans and Bridgman, 1995). Furthermore, gelsolin, which promotes depolymerization of actin filaments, has been shown to inhibit fast axonal transport in this system (Brady et al., 1984). In extruded squid axoplasm, Kuznetsov et al. (1992) observed what appeared to be the same vesicle moving along microtubules and then, subsequently, along microfilaments. Inhibitor studies provide evidence that mitochondria can move along both actin filaments and microtubules in neurons in vivo (Morris and Hollenbeck, 1995). These data support the idea that actin filament and microtubule-based transport cooperate to achieve proper organization of cellular components.The same phenomenon may be occurring in other cell types. In yeast, the mutant phenotype of the MYO2 gene, which encodes an unconventional myosin, is suppressed by overexpression of a kinesin-related protein. These two proteins are colocalized in regions of active growth where a polarized arrangement of actin plays an important role (Lillie and Brown, 1992, 1994). Microtubules are not normally required for this growth. Thus, the basis for suppression is not completely understood. However, the phenotypic suppression suggests that perhaps microtubule-based transport can substitute for actin filament–based transport, under some conditions. In polarized epithelial cells, Fath et al. (1994) have isolated a population of vesicles containing both myosin and microtubule motors. They speculate that proper transport of vesicles relies on both microtubule and actin filament–based transport.Previously, it has been shown that a class VI unconventional myosin, the Drosophila 95F unconventional myosin, transports particles along actin filaments during the syncytial blastoderm stage of Drosophila embryonic development (Mermall et al., 1994). 95F myosin activity is required for normal embryonic development (Mermall and Miller, 1995). 95F myosin is also associated with particulate structures in other cells of the embryo later in development where it may also be involved in actin-based transport. To investigate further the transport catalyzed by 95F myosin, we have begun studies to identify proteins associated with 95F myosin that might be cargoes or regulators. In this work, we have identified a protein that coimmunoprecipitates with 95F myosin. Sequence analysis reveals that this protein is the Drosophila homologue of cytoplasmic linker protein (CLIP)¹–170. CLIP-170 is believed to function as a linker between endocytic vesicles and microtubules (Pierre et al., 1992). We have named this associated protein D-CLIP-190. Colocalization of 95F myosin and D-CLIP-190 at the subcellular level at several times in development and in cultured embryonic cells provides support for the in vivo association of these two proteins. The association of a myosin and a homologue of a microtubule-binding protein suggests that these two proteins may act to coordinate the interaction between actin filaments and microtubules.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Optimization of heterologous production of the polyketide 6-MSA in Saccharomyces cerevisiae

Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes.     

Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid.

Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located.     

Adhesion molecules in subjects with COPD and healthy non-smokers: a cross sectional parallel group study

Background

The aim of the study was to investigate how the expression of adhesion molecules changes as neutrophils migrate from the circulation to the lung and if these changes differ between non-smoking subjects and smokers with and without COPD.

Methods

Non-smoking healthy subjects (n=22), smokers without (n=21) and with COPD (n=18) were included. Neutrophils from peripheral blood, sputum and bronchial biopsies were analysed for cell surface expression of adhesion molecules (CD11b, CD62L, CD162). Serum, sputum supernatant and BAL-fluid were analysed for soluble adhesion molecules (ICAM-1, -3, E-selectin, P-selectin, VCAM-1, PECAM-1).

Results

Expression of CD11b was increased on circulating neutrophils from smokers with COPD. It was also increased on sputum neutrophils in both smokers groups, but not in non-smokers, as compared to circulating neutrophils.Serum ICAM-1 was higher in the COPD group compared to the other two groups (p<0.05) and PECAM-1 was lower in smokers without COPD than in non-smoking controls and the COPD group (p<0.05). In BAL-fluid ICAM-1 was lower in the COPD group than in the other groups (p<0.05).

Conclusions

Thus, our data strongly support the involvement of a systemic component in COPD and demonstrate that in smokers neutrophils are activated to a greater extent at the point of transition from the circulation into the lungs than in non-smokers.     

Dendritic cell maturation controls adhesion, synapse formation, and the duration of the interactions with naive T lymphocytes

The initiation of adaptive immune responses requires the direct interaction of dendritic cells (DCs) with naive T lymphocytes. It is well established that the maturation state of DCs has a critical impact on the outcome of the response. We show here that mature DCs form stable conjugates with naive T cells and induce the formation of organized immune synapses. Immature DCs, in contrast, form few stable conjugates with no organized immune synapses. A dynamic analysis revealed that mature DCs can form long-lasting interactions with naive T cells, even in the absence of Ag. Immature DCs, in contrast, established only short intermittent contacts, suggesting that the premature termination of the interaction prevents the formation of organized immune synapses and full T cell activation.     

High enkephalyl peptide degradation, due to angiotensin-converting enzyme-like activity in human CSF

The metabolism of enkephalin peptides was studied in human cerebrospinal fluid. The degradation rates of (Leu)-enkephalin and (Leu)-enkephalin-Arg6 were compared and the latter was degraded at a 10-fold higher rate. The major enzyme activity was investigated by Mr determination and inhibition experiments, showing marked similarity with angiotensin-converting enzyme.