Functional analysis of dynactin and cytoplasmic dynein in slow axonal transport - PubMed (original) (raw)

MT binding of dynein and dynactin. Rat brains or radiolabeled optic nerves were removed and homogenized in PHEM buffer supplemented with phosphatase inhibitors, kinase inhibitors, and apyrase; dynactin and dynein were purified via MT affinity, followed by MgATP and NaCl release, as described in Materials and Methods. Aliquots of the supernatant and pellet fractions were resolved by 4% acrylamide and 8

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urea gel electrophoresis. HSS, A high-speed supernatant was prepared from the indicated rat tissue homogenate; either Taxol was added to the brain HSS to assemble MTs, or Taxol-stabilized MTs were added to SCb optic nerve HSS. The MTs were centrifuged through a sucrose cushion, yielding an MT-depleted supernatant (MDS) and an MT-enriched pellet (MTP). PHEM, The MT pellet was resuspended in PHEM buffer and centrifuged, yielding a supernatant (S) and an MT pellet (P). 5 mm ATP, The MT pellet was resuspended in the presence of 5 m

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MgATP and centrifuged, yielding a supernatant (S) and MT pellet (P).10 mm ATP, The MT pellet was resuspended in the presence of 10 m

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MgATP and centrifuged, yielding a supernatant (S) and MT pellet (P).1.0 m NaCl, The MT pellet was resuspended in the presence of 1.0

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NaCl and centrifuged, yielding a supernatant (S) and MT pellet (P).A, Analysis of brain dynein MT binding and elution. Portion of a gel analyzing the 530 kDa cytoplasmic dynein heavy chain (530), silver-stained. Most of the dynein from brain binds MTs in an ATP-dependent manner. B, SCb dynein binds MTs in an ATP-sensitive manner. Portion of an autoradiograph of a gel analyzing the 530 kDa cytoplasmic dynein heavy chain (530). Two sequential salt elution steps were used in this experiment, 0.6 and 1.0

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NaCl. Most (>90%) of the SCb dynein seems to bind MTs, and the majority of this (∼60%) binds in an ATP-sensitive manner. SCb dynein binding to MTs is nearly indistinguishable from whole-brain dynein binding to MTs.C, Brain p150_Glued_ dynactin binds MTs. Portion of a Western blot probed with affinity-purified UP235, which recognizes all the dynactin p150_Glued_subunit isoforms, including the p135_Glued_isoform. (This is from the same experiment as A). The p150_Glued_ and p135_Glued_isoform polypeptides are indicated (150,135). Most of the p150_Glued_isoform binds the MTs, whereas the p135_Glued_isoform does not bind the MTs. The p150_Glued_dynactin from brain binds MTs in a salt-dependent, but not ATP-dependent, manner.