ABSTRACT
A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing.
Subject(s)
Central Nervous System/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Loligo/metabolism , Nerve Tissue Proteins/metabolism , Presynaptic Terminals/metabolism , RNA-Binding Proteins/metabolism , Animals , Central Nervous System/ultrastructure , Ganglia, Invertebrate/metabolism , Ganglia, Invertebrate/ultrastructure , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/isolation & purification , Loligo/ultrastructure , Molecular Weight , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/isolation & purification , Optic Lobe, Nonmammalian/metabolism , Optic Lobe, Nonmammalian/ultrastructure , Presynaptic Terminals/ultrastructure , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/isolation & purification , Ribonucleoproteins, Small Cytoplasmic/genetics , Ribonucleoproteins, Small Cytoplasmic/metabolism , Synaptosomes/metabolism , Synaptosomes/ultrastructureABSTRACT
The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60-95%) and chick pectoralis muscle (31-83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50-60%, identities), and some also matched unconventional myosins (33-50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles.
Subject(s)
Decapodiformes/metabolism , Muscle Proteins/chemistry , Muscle, Skeletal/metabolism , Myosins/chemistry , Nerve Tissue Proteins/chemistry , Optic Lobe, Nonmammalian/metabolism , Organelles/chemistry , Protein Isoforms/chemistry , Amino Acid Sequence , Animals , Axons/chemistry , Chickens/metabolism , Microscopy, Electron , Molecular Sequence Data , Mollusca/metabolism , Muscle Proteins/isolation & purification , Muscle Proteins/ultrastructure , Myosins/isolation & purification , Myosins/ultrastructure , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/ultrastructure , Protein Isoforms/isolation & purification , Protein Isoforms/ultrastructure , Sequence Alignment , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this approximately 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.
Subject(s)
Axons/metabolism , Calmodulin/metabolism , Myosins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Ca(2+) Mg(2+)-ATPase/metabolism , Calmodulin/chemistry , Calmodulin/genetics , Cloning, Molecular , Decapodiformes , Horseshoe Crabs , Molecular Sequence Data , Organelles/metabolism , Sequence Homology, Amino AcidABSTRACT
We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.
Subject(s)
Actins/physiology , Axonal Transport/drug effects , Organelles/physiology , Adenosine Triphosphate/metabolism , Animals , Antibody Specificity , Axonal Transport/physiology , Ca(2+) Mg(2+)-ATPase/metabolism , Decapodiformes , Enzyme Activation/drug effects , Horseshoe Crabs , Immunohistochemistry , Myosins/analysis , Myosins/immunology , Organelles/enzymology , Potassium IodideABSTRACT
Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.
