Showing papers by "Worcester Foundation for Biomedical Research published in 1993"
TL;DR: This review has summarized various aspects as to how prenatal protein malnutrition affects development of the brain and has attempted to integrate several broad principles, concepts, and trends in this field in relation to findings and other studies of malnutrition insults.
648 citations
Patent•
22 Jul 1993TL;DR: In this article, the authors provided hybrid oligonucleotides having phosphorothioate or phosphorodithioate internucleotide linkages, and both deoxyribonucleosides and ribon nucleosides or 2′-substituted ribonosides with superior properties of duplex formation with RNA, nuclease resistance, and RNase H activation.
Abstract: The invention provides hybrid oligonucleotides having phosphorothioate or phosphorodithioate internucleotide linkages, and both deoxyribonucleosides and ribonucleosides or 2′-substituted ribonucleosides. Such hybrid oligonucleotides have superior properties of duplex formation with RNA, nuclease resistance, and RNase H activation.
492 citations
TL;DR: A specific role is demonstrated for dynamin and for GTP in the initial stages of receptor-mediated endocytosis in cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs.
Abstract: Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.
445 citations
TL;DR: Digital optical sectioning microscopy is performed on a highly spread NRK subclone to show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds.
Abstract: The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.
145 citations
TL;DR: The basic, proline-rich C-terminal region of dynamin is identified as the binding site for both microtubules and SH3 domains and an allosteric interaction between this region of the molecule and the N-terminals of the GTPase domain is demonstrated.
Abstract: Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potently stimulate its GTPase activity. Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to define its functional domains. Limited digestion with papain split the protein into an approximately 7- to 9-kDa microtubule-binding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end of the polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.
126 citations
Journal Article•
TL;DR: The evidence demonstrated that CYP3A enzymes catalyze N-demethylation in PB and PCN microsomes, which is difficult to reconstitute.
Abstract: Previous studies suggested that the therapeutic effect of the antimammary cancer agent tamoxifen might be related to its metabolism. This study examined the cytochrome P-450 enzymes in rat and human liver catalyzing the metabolism of tamoxifen. Incubations of tamoxifen with rat liver microsomes yielded three major polar metabolites identified as the N-oxide, N-desmethyl, and 4-hydroxy derivatives. N-Oxide formation was catalyzed by the flavin-containing monooxygenase (see part II). Carbon monoxide, SKF-525A, metyrapone, and benzylimidazole strongly inhibited N-demethylation and 4-hydroxylation, indicating the participation of P-450 monooxygenase in these reactions. Antibodies to NADPH-P450 reductase inhibited N-demethylation and 4-hydroxylation. Comparison of the metabolism of tamoxifen in untreated male and female rats demonstrated some sexual dimorphism. N-Demethylation was higher in the male rat and 4-hydroxylation was higher in the female. Treatment of rats with phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), and methylcholanthrene (MC) enhanced N-demethylation, demonstrating the potential participation of multiple P-450s in N-demethylation. Evidence strongly indicates that CYP3A enzyme(s) catalyzes N-demethylation in liver microsomes of PB- and PCN-treated rats (PB and PCN microsomes, respectively): i) N-demethylation was inhibited by cortisol and erythromycin (alternate substrates) and a time-dependent inhibition was observed with troleandomycin (TAO) in vitro; ii) treatment of female rats with TAO, followed by dissociation of the microsomal TAO-P-450 complex, elevated N-demethylation; iii) treatment of PCN-induced female rats with chloramphenicol inhibited N-demethylation; and iv) polyclonal antibodies (PAbs) to CYP3A1 inhibited N-demethylation in PCN- and PB-treated female rats. Although we were unable to reconstitute the N-demethylation activity with purified CYP3A1, which is difficult to reconstitute, collectively the evidence demonstrated that CYP3A enzymes catalyze N-demethylation in PB and PCN microsomes. By contrast, antibodies against CYP2B1/B2 did not inhibit N-demethylation and reconstituted 2B1 did not catalyze N-demethylation of tamoxifen, indicating that 2B1 was not involved. The increase in N-demethylation by MC treatment appears to be due to elevation of CYP1A1/1A2 (P-450c/d). Alternate substrates of CYP1A1/1A2 inhibited N-demethylation and reconstituted rat CYP 1A1-catalyzed N-demethylation. Surprisingly, monoclonal antibodies (MAbs) against CYP1A1/1A2 only partially inhibited, and PAbs against CYP1A1 did not inhibit N-demethylation in MC microsomes, indicating that in MC microsomes, 1A1 does not contribute significantly to that reaction. Mab anti-CYP2C11/2C6 (P-450h/k) inhibited N-demethylation in PB, PCN, and control male rat liver microsomes, suggesting that CYP2C11 and/or CYP2C6 catalyze this reaction to some extent.(ABSTRACT TRUNCATED AT 400 WORDS)
124 citations
TL;DR: Results reveal the 50-kDa polypeptide to be part of a cytosolic complex distinct from cytoplasmic dynein, however, the immunolocalization data indicate an association with microtubule minus ends, suggesting a possible interaction with cytopLasmic Dynein in the cell.
120 citations
TL;DR: In contrast with cilia and flagella, which contain numerous forms of dynein, the results are consistent with the existence of only a single cytoplasmic Dynein heavy chain gene, which appears to produce only asingle transcript.
109 citations
TL;DR: Based primarily on the observed mutant phenotypes, a function is proposed for the LARK product(s) that is consistent with the pleiotropic nature of lark mutations.
Abstract: To identify components of a circadian pacemaker output pathway, we have sought Drosophila mutations that alter the timing of eclosion but do not perturb circadian period or the expression of the activity rhythm. A mutant named lark has been identified, for which daily peaks of eclosion occur abnormally early while populations are synchronized to either light/dark or temperature cycles. The temporal phasing of locomotor activity in lark mutants, however, is entirely normal, as is the free-running period of the circadian pacemaker. The lark strain carries a single P-element insertion which, interestingly, has a dominant effect on the timing of eclosion, but is also associated with a recessive embryonic lethal phenotype. The analysis of excision-generated alleles suggests that the lark gene encodes an essential function. This function is apparently mediated by a transcription unit that is interrupted by the P-induced lark mutation. A combination of in situ hybridization analysis and reporter (beta-gal) staining indicates that this transcription unit expresses mRNAs throughout the embryonic central nervous system and in a defined subset of cells in the nervous system of pharate adults. RNAs are first detected at about embryonic stage 11, just prior to the stage at which lethality occurs in lark homozygotes. Based primarily on the observed mutant phenotypes, a function is proposed for the LARK product(s) that is consistent with the pleiotropic nature of lark mutations.
104 citations
Journal Article•
TL;DR: Tamoxifen-N-oxide was found to be readily reduced to tamox ifen by rat or human liver microsomes, in the presence of NADPH; the extent of reduction was dramatically increased when incubations were supplemented with methimazole, suggesting that the N-oxide may serve as a storage form for tamoxIFen in vivo.
Abstract: Earlier studies demonstrated that the major metabolites of tamoxifen generated by mammalian liver microsomes are the corresponding N-oxide, N-desmethyl, and 4-hydroxy derivatives. This study examines the enzymatic activity catalyzing the formation of tamoxifen N-oxide by mammalian liver microsomes. Incubations of tamoxifen with liver microsomes from the various species, supplemented with NADPH, yielded the N-oxide, N-desmethyl, and 4-hydroxy derivatives. Inhibition of N-oxide accumulation by mild heat and low concentrations of methimazole in rat liver microsomes indicated that this reaction is catalyzed by the flavin-containing monooxygenase (FMO). Antibodies to NADPH-P-450 reductase inhibited N-demethylation and 4-hydroxylation, but not N-oxidation, supporting the aforementioned conclusion. Purified mouse liver microsomal FMO converted tamoxifen solely into the N-oxide, providing direct evidence for FMO involvement. Human liver microsomes formed the same tamoxifen metabolites, albeit at a much lower rate. Inhibitors of FMO diminished the accumulation of N-oxide by human liver microsomes, indicating involvement of FMO. Tamoxifen-N-oxide was found to be readily reduced to tamoxifen by rat or human liver microsomes, in the presence of NADPH; the extent of reduction was dramatically increased when incubations were supplemented with methimazole. The facile reduction of tamoxifen N-oxide back to tamoxifen suggests that the N-oxide may serve as a storage form for tamoxifen in vivo.
99 citations
TL;DR: The results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins.
Abstract: Although the distribution of filamentous actin is well characterized in many cell types, the distribution of nonfilamentous actin remains poorly understood. To determine the relative distribution of filamentous and nonfilamentous actin in cultured NRK cells, we have used a number of labeling agents that differ with respect to their specificities toward the filamentous or nonfilamentous form, including monoclonal and polyclonal anti-actin antibodies, vitamin D-binding protein (DBP), and fluorescent phalloidin. Numerous punctate structures were identified that bind poorly to phalloidin but stain positively with several anti-actin antibodies. These bead structures also stain with DBP, suggesting that they are enriched in nonfilamentous actin. Similar punctate structures were observed after the microinjection of fluorescently labeled actin into living cells, allowing us to examine their dynamics in living cells. The actin-containing punctate structures were observed predominantly in the region behind lamellipodia, particularly in spreading cells induced by wounding confluent monolayers. Time-lapse recording of cells injected with fluorescent actin indicated that they form continuously near the leading edge and move centripetally toward the nucleus. Our results suggest that at least part of the unpolymerized actin molecules are localized at discrete sites, possibly as complexes with monomer sequestering proteins. These structures may represent transient storage sites of G-actin within the cell which can be transformed rapidly into actin filaments upon stimulation by specific signals.
TL;DR: While the effect of 5-HT on the S-K+ channel is mediated exclusively by cAMP, the effect on the Ca2+ current can be mimicked by phorbol 12,13- dibutyrate (PDBu) as well as by intracellular injection of cAMP.
Abstract: In the sensory neurons of Aplysia, 5-HT acts through cAMP to reduce current flow through two classes of K+ channels, the S-K + channel and a transient K+ channel (Ikv). In addition, 5-HT increases a voltage-dependent, nifedipine-sensitive Ca2+ current. In this article we show that, while the effect on the S-K+ channel is mediated exclusively by cAMP, the effect on the Ca2+ current can be mimicked by phorbol 12,13-dibutyrate (PDBu) as well as by intracellular injection of cAMP. We then use specific blockers of protein kinase C (PKC) and the cAMP-dependent protein kinase A (PKA) to examine the roles of PKC and PKA in mediating the effect of 5-HT on the nifedipine-sensitive Ca2+ current. We find that H-7, a kinase inhibitor that appears to inhibit PKC more effectively than PKA in intact Aplysia neurons, reverses the increase in the Ca2+ current produced by PDBu. Moreover, H-7 partially blocks the effect of 5-HT on the Ca2+ current without affecting the decrease in the S-K+ current. A more specific PKC inhibitor (the 19-31 pseudosubstrate of PKC) also partially blocks the increase in the Ca2+ current produced by 5-HT, suggesting that this increase is mediated by PKC. Rp-cAMPS, a specific blocker of PKA, did not block the increase in the Ca2+ current produced by 5-HT, suggesting that the effect of 5-HT on this current may be mediated to only a small extent by PKA. The effect of 5-HT on the S-K+ current and the Ca2+ current can also be separated on basis of the time course of their appearance. The fact that the decrease in the S-K+ current precedes the increase in Ca2+ current suggests that there may be a temporal difference in the activation of the two kinase systems.
TL;DR: The results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation, which is required for their binding to adenovirus and human beta-globin pre-mRNAs.
Abstract: The C hnRNP proteins bind to nascent pre-mRNA and are thought to participate in an early step of the pre-mRNA splicing pathway. We report here that C hnRNP proteins are phosphorylated by a casein kinase II activity in a HeLa cell nuclear extract and that dephosphorylation of C hnRNP proteins is inhibited by the specific protein-serine/threonine-phosphatase 1/2A inhibitor okadaic acid. We further find that dephosphorylation of C hnRNP proteins is required for their binding to adenovirus and human beta-globin pre-mRNAs. These results indicate that the participation of C hnRNP proteins in pre-spliceosome assembly is coupled to a dynamic cycle of their phosphorylation and dephosphorylation.
TL;DR: Molecular analysis of several of the polypeptide components of dynein and a related complex has provided important new insight into their structural organization and mechanism of action in the cell.
Abstract: Dynein is a large enzyme complex that has been found in recent years to be responsible for a variety of forms of intracellular movement associated with microtubules. Molecular analysis of several of the polypeptide components of dynein and a related complex has provided important new insight into their structural organization and mechanism of action in the cell.
TL;DR: A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis, and this strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response.
Abstract: A new mutant strain of Chlamydomonas, ptx1, has been identified which is defective in phototaxis This strain swims with a rate and straightness of path comparable with that of wild-type cells, and retains the photoshock response Thus, the mutation does not cause any gross defects in swimming ability or photoreception, and appears to be specific for phototaxis Calcium is required for phototaxis in wild-type cells, and causes a concentration-dependent shift in flagellar dominance in reactivated, demembranated cell models ptx1-reactivated models are defective in this calcium-dependent shift in flagellar dominance This indicates that the mutation affects one or more components of the calcium-dependent axonemal regulatory system, and that this system mediates phototaxis The reduction or absence of two 75-kD axonemal proteins correlates with the nonphototactic phenotype Axonemal fractionation studies, and analysis of axonemes from mutant strains with known structural defects, failed to reveal the structural localization of the 75-kD proteins within the axoneme The proteins are not components of the outer dynein arms, two of the three types of inner dynein arms, the radial spokes, or the central pair complex Because changes in flagellar motility ultimately require the regulation of dynein activity, cell models from mutant strains defective in specific dynein arms were reactivated at various calcium concentrations Mutants lacking the outer arms, or the I1 or I2 inner dynein arms, retain the wild-type calcium-dependent shift in flagellar dominance Therefore, none of these arms are the sole mediators of phototaxis
TL;DR: Four splice variants of the NMDAR1 subunit are expressed in Xenopus oocytes, and it is found that homomeric assemblies of this subunit, in the absence of other subunits, exhibit ethanol sensitivity comparable to that seen in neurons.
TL;DR: This review discusses agrin's role in orchestrating the molecular topography of the post-synaptic apparatus at nerve-muscle synapses and the emerging evidence suggesting a role for agrin in synaptogenesis in the brain.
TL;DR: A role for muscle agrin in neuromuscular junction development is suggested by using species-specific anti-agrin antibodies to show that Muscle agrin colocalizes with nerve-induced AChR clusters in chimeric nerve- muscle cocultures.
Abstract: The synapse-organizing protein agrin is expressed by muscle cells. In this study we begin to characterize the role of muscle agrin in synapse formation by investigating its distribution and expression during the formation of nerve-induced ACh receptor (AChR) aggregates in vitro. We have used species-specific anti-agrin antibodies to show that muscle agrin colocalizes with nerve-induced AChR clusters in chimeric nerve- muscle cocultures. Furthermore, quantitation by radioimmune assay shows that the expression of muscle agrin by cultured chick myotubes is increased more than twofold by coculture with cells from the motoneuron- rich ventral spinal cord. These data suggest a role for muscle agrin in neuromuscular junction development.
TL;DR: The findings suggest that the integrity of the central loop is of little significance for oligomer formation but that it is more important for the final step in pore assembly or alternatively for determining the correct structure of the conductive channel.
31 Dec 1993
TL;DR: This work has shown that the spindle structure established as it forms during prometaphase ensures that the replicated chromosomes are segregated equivalently to two daughter cells, which is a major goal of cell division research.
Abstract: Chromosome distribution during both mitosis and meiosis is effected by the “spindle”, a complex ensemble formed from an interaction between chromosomes and microtubules (MTs). One of the most important characteristics of the spindle is its bipolar structure, established as it forms during prometaphase, which ensures that the replicated chromosomes are segregated equivalently to two daughter cells. A major goal of cell division research is to understand the mechanism of spindle morphogenesis and how bipolarity is established.
TL;DR: The results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly.
Abstract: Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.
TL;DR: Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences A ChR levels in developing neurons in situ.
Abstract: Chick ciliary ganglion neurons receive innervation from a single source, the accessory oculomotor nucleus (AON), and nicotinic ACh receptors (AChRs) mediate chemical synaptic transmission through the ganglion. Previous experiments examining the developmental expression of AChRs in embryonic chick ciliary ganglion neurons in situ have shown that AChR levels increase substantially in the neurons at the time of innervation. Prior to synapse formation, few AChRs are detected in the neurons. In the present experiments, the role of presynaptic inputs in inducing an increase in AChRs was established by examining AChR levels in ciliary ganglion neurons that have been deprived of innervation by surgical ablation of the AON prior to synapse formation. AChR levels were dramatically reduced in neurons of input-deprived ganglia as compared to control innervated neurons at all developmental stages examined from embryonic day (ED) 5 to ED 12 as determined by indirect immunocytochemical labeling of frozen ganglion sections with the anti- AChR monoclonal antibody mAb 35, and light microscopy. In contrast, neuronal somata of input-deprived and control ganglia had equivalent levels of immunolabeling for three other components, a transmembrane glycoprotein of synaptic vesicles, SV2, and two microtubule-associated proteins, MAP 1B and MAP 2, from ED 5 up to ED 10. The results demonstrate that presynaptic inputs specifically increase the levels of AChR expression in developing neurons. In addition, changes in the levels of immunolabeling for AChRs, SV2, MAP 1B, and MAP 2 in neuronal somata after ED 10 demonstrate that other major developmental events also influence the levels of these components in neurons. Declines in the intensity of AChR, SV2, MAP 1B, and MAP 2 immunolabeling within a subset of neuronal somata in both operated and control ganglia at ED 10 and 12 coincide with the period of neuronal cell death. Increases in AChR labeling in the rest of the neuronal population of input-deprived ganglia at ED 12 suggest that, in addition to innervation, synapse formation with the peripheral target tissue influences AChR levels in developing neurons in situ.
TL;DR: It is proposed that caltrin is a regulator of sperm signal transduction pathways activated by zona pellucida binding, a previously characterized regulator of Ca2+ transport in bovine sperm.
TL;DR: The results indicate that the biological activity of agrin is mediated through intracellular signal transduction events triggered by ligand binding to the agrin receptor.
TL;DR: Conurrent radioisotopic flux assays confirm that calcium influx is, indeed, increased in the chronically treated cells, but acute exposure to ethanol at the same concentration as those used for the chronic studies greatly reduced the magnitude of the currents and net calcium influx.
TL;DR: The actions of halothane on a diverse collection of voltage-gated potassium channels expressed in Xenopus oocytes are examined, and it is found that they are all sensitive at clinically relevant concentrations.
TL;DR: The results show that the reproductive capacity of maternal centrosomes is degraded during meiosis I, not during oogenesis, and that the cytoplasmic conditions which eliminate this reproductive capacity are no longer active once the zygote has entered the first mitotic cell cycle.
01 Jan 1993
TL;DR: A critical role for dynamin in the early stages of endocytosis is suggested as a result of molecular cloning of rat brain dynamin, which revealed sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product the yeast mitochondrial replication gene MGM1.
Abstract: Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.