Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.

S P Gilbert; R D Sloboda

doi:10.1083/jcb.99.2.445

Bidirectional transport of fluorescently labeled vesicles introduced into extruded axoplasm of squid Loligo pealei.

The Journal of Cell Biology ◽

10.1083/jcb.99.2.445 ◽

1984 ◽

Vol 99 (2) ◽

pp. 445-452 ◽

Cited By ~ 27

Author(s):

S P Gilbert ◽

R D Sloboda

Keyword(s):

Axonal Transport ◽

Giant Axon ◽

Epifluorescence Microscopy ◽

Differential Interference Contrast Microscopy ◽

Fast Axonal Transport ◽

Bidirectional Transport ◽

Interference Contrast Microscopy ◽

The Mean ◽

Loligo Pealei ◽

Vesicle Movement

A reconstituted model was devised to study the mechanisms of fast axonal transport in the squid Loligo pealei. Axonal vesicles were isolated from axoplasm of the giant axon and labeled with rhodamine-conjugated octadecanol, a membrane-specific fluorescent probe. The labeled vesicles were then injected into a fresh preparation of extruded axoplasm in which endogenous vesicle transport was occurring normally. The movement of the fluorescent, exogenous vesicles was observed by epifluorescence microscopy for as long as 5 min without significant photobleaching, and the transport of endogenous, nonfluorescent vesicles was monitored by video-enhanced differential interference-contrast microscopy. The transport of fluorescent, exogenous vesicles was shown to be bidirectional and ATP-dependent and occurred at a mean rate of 6.98 +/- 4.11 micron/s (mean +/- standard deviation, n = 41). In comparison, the mean rate of transport of nonfluorescent, endogenous vesicles in control axoplasm treated with vesicle buffer alone was 4.76 +/- 1.60 micron/s (n = 64). These rates are slightly higher than the mean rate of endogenous vesicle movement in extruded axoplasm (3.56 +/- 1.05 micron/s, n = 40) not subject to vesicles or vesicle buffer. Not all vesicles and organelles, exogenous or endogenous, were observed to move. In experiments in which proteins of the surface of the fluorescent vesicles were digested with trypsin before injection, no movement of the fluorescent vesicles was observed, although the transport of endogenous vesicles and organelles appeared to proceed normally. The results summarized above indicate that isolated vesicles, incorporated into axoplasm, move with the characteristics of fast axonal transport. Because the vesicles are fluorescent, they can be readily distinguished from nonfluorescent, endogenous vesicles. Moreover, this system permits vesicle characteristics to be experimentally manipulated, and therefore may prove valuable for the elucidation of the mechanisms of fast axonal transport.

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Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy.

The Journal of Cell Biology ◽

10.1083/jcb.103.5.1921 ◽

1986 ◽

Vol 103 (5) ◽

pp. 1921-1931 ◽

Cited By ~ 146

Author(s):

D J Goldberg ◽

D W Burmeister

Keyword(s):

Axonal Transport ◽

Growth Cone ◽

Axon Growth ◽

Leading Edge ◽

Main Body ◽

Differential Interference Contrast ◽

Differential Interference Contrast Microscopy ◽

Fast Axonal Transport ◽

Contrast Microscopy ◽

Interference Contrast Microscopy

The regenerative growth in culture of the axons of two giant identified neurons from the central nervous system of Aplysia californica was observed using video-enhanced contrast-differential interference contrast microscopy. This technique allowed the visualization in living cells of the membranous organelles of the growth cone. Elongation of axonal branches always occurred through the same sequence of events: A flat organelle-free veil protruded from the front of the growth cone, gradually filled with vesicles that entered by fast axonal transport and Brownian motion from the main body of the growth cone, became more voluminous and engorged with organelles (vesicles, mitochondria, and one or two large, irregular, refractile bodies), and, finally, assumed the cylindrical shape of the axon branch with the organelles predominantly moving by bidirectional fast axonal transport. The veil is thus the nascent axon. Because veils appear to be initially free of membranous organelles, addition of membrane to the plasmalemma by exocytosis is likely to occur in the main body of the growth cone rather than at the leading edge. Veils almost always formed with filopodial borders, protruding between either fully extended or growing filopodia. Therefore, one function of the filopodia is to direct elongation by demarcating the pathway along which axolemma flows. Models of axon growth in which the body of the growth cone is pulled forward, or in which advance of the leading edge is achieved by filopodial shortening or contraction against an adhesion to the substrate, are inconsistent with our observations. We suggest that, during the elongation phase of growth, filopodia may act as structural supports.

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Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.3.c929 ◽

1996 ◽

Vol 271 (3) ◽

pp. C929-C943 ◽

Cited By ~ 49

Author(s):

S. F. Hamm-Alvarez ◽

X. Wei ◽

N. Berndt ◽

M. Runnegar

Keyword(s):

Immunofluorescence Microscopy ◽

Maximum Dose ◽

Protein Phosphatases ◽

Differential Interference Contrast Microscopy ◽

Contrast Microscopy ◽

Interference Contrast Microscopy ◽

Microscopy Analysis ◽

High Doses ◽

Vesicle Movement ◽

Cellular Components

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

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Modeling Bidirectional Transport of New and Used Organelles in Fast Axonal Transport in Neurons

Journal of Heat Transfer ◽

10.1115/1.4002304 ◽

2010 ◽

Vol 133 (1) ◽

Cited By ~ 2

Author(s):

A. V. Kuznetsov

Keyword(s):

Axonal Transport ◽

Cell Body ◽

Molecular Motors ◽

Molecular Motor ◽

Fast Axonal Transport ◽

Concentration Difference ◽

Neuron Body ◽

Microtubule Polarity ◽

Bidirectional Transport ◽

The Right

This paper develops a model for simulating transport of newly synthesized material from the neuron body toward the synapse of the axon as well as transport of misfolded and aggregated proteins back to the neuron body for recycling. The model demonstrates that motor-assisted transport, much similar to diffusion, can occur due to a simple concentration difference between the cell body and the synapse; organelles heading to the synapse do not need to attach preferably to plus-end-directed molecular motors, same as organelles heading to the neuron body for recycling do not need to attach preferably to minus-end-directed molecular motors. The underlying mechanics of molecular-motor-assisted transport is such that organelles would be transported to the right place even if new and used organelles had the same probability of attachment to plus-end-directed (and minus-end-directed) motors. It is also demonstrated that the axon with organelle traps and a region with a reversed microtubule polarity would support much smaller organelle fluxes of both new and used organelles than a healthy axon. The flux of organelles is shown to decrease as the width of organelle traps increases.

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Fast axonal transport in extruded axoplasm from squid giant axon

Science ◽

10.1126/science.6183745 ◽

1982 ◽

Vol 218 (4577) ◽

pp. 1129-1131 ◽

Cited By ~ 208

Author(s):

S. Brady ◽

R. Lasek ◽

R. Allen

Keyword(s):

Axonal Transport ◽

Giant Axon ◽

Squid Giant Axon ◽

Fast Axonal Transport

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Transport of ER vesicles on actin filaments in neurons by myosin V

Journal of Cell Science ◽

10.1242/jcs.111.21.3221 ◽

1998 ◽

Vol 111 (21) ◽

pp. 3221-3234 ◽

Cited By ~ 9

Author(s):

J.S. Tabb ◽

B.J. Molyneaux ◽

D.L. Cohen ◽

S.A. Kuznetsov ◽

G.M. Langford

Keyword(s):

Axonal Transport ◽

Actin Filaments ◽

Giant Axon ◽

Vesicle Transport ◽

Amino Acid Sequences ◽

Dependent Manner ◽

Tail Domain ◽

Myosin V ◽

Fast Axonal Transport

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was tightly bound and not extracted during isolation. These vesicles were identified as smooth ER by use of an antibody to an ER-resident protein, ERcalcistorin/protein disulfide isomerase (EcaSt/PDI). Furthermore, an antibody to squid myosin V was used in immunogold EM studies to show that myosin V localized to these vesicles. The antibody was generated to a squid brain myosin (p196) that was classified as myosin V based on comparisons of amino acid sequences of tryptic peptides of this myosin with those of other known members of the myosin V family. Dual labeling with the squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. Finally, antibody inhibition experiments were performed with two myosin V-specific antibodies to show that myosin V motor activity is required for transport of vesicles on actin filaments in axoplasm. One antibody was made to a peptide in the globular tail domain and the other to the globular head fragment of myosin V. Both antibodies inhibited vesicle transport on actin filaments by greater than 90% compared to controls. These studies provide the first direct evidence that ER vesicles are transported on actin filaments by myosin V. These data confirm the role of actin filaments in fast axonal transport and provide support for the dual filament model of vesicle transport.

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