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Methods in Cell
Biology
The Neuronal Cytoskeleton,
Motor Proteins, and Organelle
Trafficking in the Axon
Volume 131
Series Editors
Leslie Wilson
Department of Molecular, Cellular and Developmental Biology
University of California
Santa Barbara, California
Phong Tran
University of Pennsylvania
Philadelphia, USA &
Institut Curie, Paris, France
Methods in Cell
Biology
The Neuronal Cytoskeleton,
Motor Proteins, and Organelle
Trafficking in the Axon
Volume 131
Edited by
K. Kevin Pfister
Department of Cell Biology, Charlottesville, USA
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Contributors
Stefanie Alber
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
C.J. Alexander
Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National
Institutes of Health, MD, USA
Adam W. Avery
Department of Genetics, Cell Biology, and Development, University of Minnesota,
Minneapolis, MN, USA
Peter W. Baas
Department of Neurobiology and Anatomy, Drexel University College of Medicine,
Philadelphia, PA, USA
Alexandre D. Baffet
Department of Pathology and Cell Biology, Columbia University, New York,
NY, USA
Lisa Baker
Marine Biological Laboratory, Woods Hole, MA, USA
Gary Banker
Jungers Center for Neurosciences Research, Oregon Health and Science
University, Portland, OR, USA
Marvin Bentley
Mark M. Black
Department of Anatomy and Cell Biology, Temple University School of Medicine,
Kiev R. Blasier
Department of Cell Biology, University of Virginia, Charlottesville, VA, USA
Scott T. Brady
Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy
and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA
Anthony Brown
Department of Neuroscience, The Ohio State University, Columbus, OH, USA
xiii
xiv Contributors
Kristy J. Brown
Research Center for Genetic Medicine, Children’s National Health System,
Washington, DC, USA; Department of Integrative Systems Biology, Institute of
Biomedical Sciences, The George Washington University, Washington, DC, USA
Alma L. Burlingame
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF,
San Francisco, CA, USA
John C. Cain
Aurélie Carabalona
NY, USA
Anaël Chazeau
Cell Biology, Department of Biology, Faculty of Science, Utrecht University,
Utrecht, The Netherlands
Michael Chein
Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and
the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
Tiago J. Dantas
NY, USA
David D. Doobin
NY, USA
Ella Doron-Mandel
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot,
Israel
Catherine M. Drerup
Department of Cell, Developmental and Cancer Biology, School of Medicine,
Oregon Health & Science University, Portland, OR, USA
Noelle D. Dwyer
Department of Cell Biology, University of Virginia School of Medicine,
Charlottesville, VA, USA
Mike Fainzilber
Department of Biological Chemistry, Weizmann Institute of Science,
Rehovot, Israel
Contributors xv
J. Daniel Fenn
Xiaoqin Fu
Center for Neuroscience Research, Children’s National Health System,
Washington, DC, USA
Kathlyn J. Gan
Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, BC, Canada
Archan Ganguly
Department of Pathology, University of California, San Diego, La Jolla, CA, USA
Shani Gluska
J.A. Hammer, III
Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National
Institutes of Health, MD, USA
Thomas S. Hays
Erika L.F. Holzbaur
Department of Physiology, University of Pennsylvania Perelman School of
Medicine, Philadelphia, PA, USA; Neuroscience Graduate Group, University of
Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Casper C. Hoogenraad
Daniel J. Hu
NY, USA
Chung-Fang Huang
University, Portland, OR, USA; National Laboratory Animal Center, NARLabs,
Taipei, Taiwan
Ariel Ionescu
xvi Contributors
Kerstin M. Janisch
Department of Cell Biology, University of Virginia School of Medicine,
Minsu Kang
Marine Biological Laboratory, Woods Hole, MA, USA; Department of Anatomy
and Cell Biology, University of Illinois at Chicago, Chicago, IL, USA
Lukas C. Kapitein
Eugene A. Katrukha
Noopur V. Khobrekar
NY, USA
Eva Klinman
Kelsey Ladt
Department of Neurosciences, University of California, San Diego, La Jolla,
CA, USA
Zofia M. Lasiecka
Children’s National Medical Center, Washington, DC, USA
Seung Joon Lee
Department of Biological Sciences, University of South Carolina, Columbia,
SC, USA
Lanfranco Leo
Min-gang Li
Judy S. Liu
Center for Neuroscience Research, Children’s National Health System,
Washington, DC, USA
Contributors xvii
James B. Machamer
Department of Neurology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Katalin F. Medzihradszky
David J. Mitchell
Paula C. Monsma
Gerardo Morfini
Department of Anatomy and Cell Biology, University of Illinois at Chicago,
Chicago, IL, USA; Marine Biological Laboratory, Woods Hole, MA, USA
Kanneboyina Nagaraju
Alex V. Nechiporuk
Department of Cell, Developmental and Cancer Biology, School of Medicine,
Oregon Health & Science University, Portland, OR, USA
Amanda L. Neisch
Jeffrey J. Nirschl
Juan A. Oses
Eran Perlson
K. Kevin Pfister
xviii Contributors
Sree Rayavarapu
Mitchell W. Ross
Nimrod Rotem
Subhojit Roy
Department of Neurosciences, University of California, San Diego, La Jolla, CA,
USA; Department of Pathology, University of California, San Diego, La Jolla, CA,
USA
Philipp Schätzle
Cell Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
Michael A. Silverman
Department of Molecular Biology and Biochemistry, Simon Fraser University,
Burnaby, BC, Canada; Department of Biological Sciences, Simon Fraser
University, Burnaby, BC, Canada; Brain Research Centre, University of British
Columbia, Vancouver, BC, Canada
Yuyu Song
Marine Biological Laboratory, Woods Hole, MA, USA; Yale School of Medicine,
Department of Genetics and Howard Hughes Medical Institute, Boyer Center,
New Haven, CT, USA
Jeffery L. Twiss
Department of Biological Sciences, University of South Carolina, Columbia,
SC, USA
Atsuko Uchida
Richard B. Vallee
NY, USA
Bettina Winckler
Department of Neuroscience, University of Virginia Medical School,
Contributors xix
Rui Yang
Julie Yi
NY, USA
Wenqian Yu
Jie Zhou
NY, USA
Preface
Investigations into fundamental questions in cell biology have long benefited from
experiments that utilize neuronal systems. Neurons have proven particularly useful
model systems for enhancing our understanding of intracellular transport. Their long
thin axons, which can comprise 95% of the cellular volume, cannot be maintained by
diffusion alone, and thus they may be regard as specialized for transport. In addition,
their morphology makes axons ideal systems to image motor protein-based move-
ment with live cell microscopy. These properties render axonal transport an effective
model for investigating the cytoskeleton, motor proteins, and organelle transport.
The chapters in this volume describe methods that utilize live cell imagining,
genetic, molecular, biochemical, and proteomic approaches in neuronal systems to
characterize and explore fundamental questions related to intracellular motility,
especially axonal transport. The contributors employ a wide variety of culture
systems including sympathetic, cortical, hippocampal, dorsal root ganglion, and
Purkinje neurons as well as in vitro slice cultures and axoplasm from the squid giant
axon; as well as model organisms Drosophila, zebrafish, and mice. The first chapter
introduces the basic paradigm for the mechanism(s) of movement in the axon. It
reviews in vivo pulse labeling experiments which identified the movement of three
distinct sets of structural components from the cell body down the axon; membrane-
bounded organelles, microtubule, and neurofilaments, and actin with the over 200
remaining axonal proteins. The chapter continues by discussing recent live cell
imaging data, utilizing the excellent optical properties of long thin axons, to define
the mechanisms for the moment of the structures. The volume is then organized into
three overlapping areas with methods chapters that focus on (1) cytoskeletal protein
dynamics and filament transport, (2) the motor proteins responsible for transport,
and (3) the transport of membrane-bounded organelle cargos.
Procedures are given for the live imaging of neurofilament transport and actin
dynamics and transport in cultured neurons. In addition, methods are described to
image tubulin dynamics in cultured hippocampal slices and single molecule resolu-
tion of tubulin and microtubule plus-end-tracking proteins in cultured neurons.
Techniques for live imaging of the movement of cytoplasmic dynein and the initia-
tion of retrograde organelle transport in axons of cultured neurons are also
presented. Assays to probe kinesin motor domain function and the role of a kinesin
family member in cytokinesis in neuroprogenitors are reviewed. Genetic and imag-
ing approaches to analyze motor protein function and organelle motility and neuro-
progenitor migration are provided using zebrafish, Drosophila, and mouse models.
A variety of approaches to image and analyze membrane-bounded organelle and
other cargo motility (including endosomes, lysosomes, autophagosomes, mitochon-
dria, signaling endosomes, viruses, and ribonucleoprotein particles) in axons, den-
drites, and squid axoplasm are discussed. These include utilizing microfluidics
chambers for culturing neurons; labeling the membrane-bounded organelle cargos
with dyes or fluorescent-tagged proteins; tracking internalized transmembrane
xxi
xxii Preface
proteins with quantum dot- or fluorochrome-labeled ligands or antibodies; and

investigating effect of the Alzheimer’s disease peptide b-amyloid on organelle
transport.
Several chapters take advantage of molecular and biochemical methods to
analyze cytoskeletal and motor protein activity. The squid axoplasm system is
utilized to investigate kinase pathways of phosphorylation of filament subunits
and motor proteins. A proteomics method is presented to probe the effects of mouse
mutations on the cytoskeleton and motor proteins; and affinity chromatography is
used to investigate motor proteins association with ribonucleoprotein particle trans-
port in axons. Two contributions discuss methods for knocking down the expression
of neuronal proteins using RNAi, one focuses on using siRNA in sympathetic and
hippocampal neurons; the second describes a plasmid-based approach to reduce
myosin Va levels in cultured Purkinje cells.
CHAPTER
Axonal transport: The

orderly motion of axonal
structures 1
Mark M. Black
Department of Anatomy and Cell Biology,
Temple University School of Medicine, Philadelphia, PA, USA
E-mail: mark.black@temple.edu
CHAPTER OUTLINE
1. Pulse-Labeling Studies of Axonal Transport ............................................................. 2
2. Live-Cell Imaging of Axonal Transport...................................................................... 7
2.1 FC and the Movement of Vesicular Cargoes................................................ 7
2.2 Slow Axonal Transport and the Movement of Cytoskeletal Polymers ............. 8
2.3 Neurofilaments are Transported in Axons................................................... 8
2.4 Microtubules and Slow Axonal Transport ................................................. 10
2.5 SCb and the Movement of Soluble Proteins of Axoplasm........................... 12
3. Summary ............................................................................................................. 15
References ............................................................................................................... 15
Abstract
Axonal transport is a constitutive process that supplies the axon and axon terminal with
materials required to maintain their structure and function. Most materials are supplied
via three rate components termed the fast component, slow component a, and slow
component b. Each of these delivers a distinct set of materials with distinct transport
kinetics. Understanding the basis for how materials sort among these rate components
and the mechanisms that generate their distinctive transport kinetics have been long-
standing goals in the field. An early view emphasized the relationships between axonally
transported cargoes and cytological structures of the axon. In this article, I discuss key
observations that led to this view and contemporary studies that have demonstrated its
validity and thereby advanced the current understanding of the dynamics of axonal
structure.
Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.001 1

© 2016 Elsevier Inc. All rights reserved.
2 CHAPTER 1 Axonal transport: The orderly motion of axonal structures
Axonal transport is the process by which proteins and other materials synthe-
sized in the neuronal cell body are delivered to the axon and axon terminal.
This is a constitutive process that occurs throughout the life of neurons, supply-
ing axons with materials needed to maintain their structure and function. The
notion that the axon depends on the cell body dates back to the nineteenth cen-
tury, based on the observation that axons disconnected from their cell bodies
degenerate (Ramon y Cajal, 1928). However, it was not until 1948 that movement
of materials in axons was first revealed by Weiss and Hiscoe, who partially con-
stricted axons and observed that axoplasm accumulated immediately proximal to
the constriction, suggesting a proximal-to-distal movement of axonal materials.
Upon release of the constriction, the accumulated axoplasm moved anterogradely
at z1 mm/day, thus identifying what later came to be known as slow axonal
transport.
Since this pioneering work, axonal transport has been studied extensively with
two experimental approaches providing most of the current understanding. One
uses radioactive precursors to pulse-label axonally transported materials and the
other uses imaging techniques to directly observe transport in living axons. These
two approaches provide distinct but complementary information (Brown, 2009).
Pulse-chase approaches provide indirect information on movement of materials in
axons in intact animals over time scales of hours to months whereas live-cell imag-
ing directly visualizes axonal transport over time frames of seconds to hours. Below,
I discuss contributions of these approaches to the current understanding of the
cargoes that undergo axonal transport and their transport behavior as seen at short
and long time scales.
1. PULSE-LABELING STUDIES OF AXONAL TRANSPORT

The pulse-labeling approach has revealed the kinetics of protein transport in axons
over long time scales and the identity of many transported proteins. Typically, radio-
active amino acids are injected into the environment surrounding the neuron cell
bodies under study. The amino acids are taken into the neurons and incorporated
into proteins, some of which are then transported into their axons. Because the
amino acids are cleared relatively rapidly by the circulation, this procedure produces
a pulse of labeling in vivo. To visualize the transport of the pulse-labeled proteins,
the nerve containing them is cut into consecutive pieces of a few millimeters in
length and the distribution of radioactivity along its length quantified. Also, the iden-
tity of specific radioactive proteins in the nerve segments has been determined using
biochemical procedures. As each animal provides a single time point for analysis,
multiple animals must be examined, each at different times after labeling.
Comparing the results at the various times yields a detailed, though indirect, picture
of the movement of proteins in axons.
This approach has been used with a variety of organisms and the essential
results obtained are consistent among systems. The transported pulse-labeled
1. Pulse-Labeling studies of axonal transport 3
proteins are distributed along the axons as waves with distinct crests and fronts
(Figure 1(A)). The positions and shapes of the waves change as a function of
time after injection based on the transport behavior of the proteins. At time frames
of hours, waves of pulse-labeled proteins are seen that advance at z50e400 mm/
day (0.6e5 mm/s) (reviewed in Grafstein & Forman, 1980). This corresponds to the
fast component (FC) of axonal transport. FC has both anterograde (soma toward
axon tip) and retrograde (axon tip toward soma) components. There is also a
slow component which moves at average rates of 0.2e10 mm/day (0.0002e
0.1 mm/s). Slow axonal transport consists of two subcomponents, slow component
a (SCa) and slow component b (SCb), that differ in specific protein composition and
transport rate. SCa moves at modal rates of 0.2e3 mm/day, while SCb moves at
2.0e10 mm/day (the range in rates reflects variations among different populations
of neurons). These three rate components provide most of the materials delivered to
the axon by axonal transport.
Cell fractionation and electron microscopic autoradiographic studies (Di
Giamberardino, Bennett, Koenig, & Droz, 1973; Droz, Koenig, Biamberardino, &
Di Giamberardino, 1973; Lorenz & Willard, 1978) showed that fast and slow axonal
transport deliver distinct materials to the axon. This result was confirmed by gel elec-
trophoretic analyses of the proteins comprising FC, SCa, and SCb (Tytell, Black,
Garner, & Lasek, 1981; Willard, Cowan, & Vagelos, 1974). FC and SCb each consists
of hundreds of proteins, whereas SCa transports comparatively few, and strikingly
very few proteins are present in more than one rate component (Figure 1(B) and
(C)). Thus, the underlying mechanisms of axonal transport prevent the mixing of pro-
teins as they move past each other in the axon. The structural hypothesis of axonal
transport was put forth to explain this and other differences between FC, SCa, and
SCb (Lasek, 1980; Lasek, Garner & Brady, 1984). This hypothesis posits that pro-
teins are actively transported in the axon either as integral parts of moving cytological
structures or in association with these structures. At the time, the strongest support
was for FC for which multiple criteria showed was associated with membrane-bound
organelles (Dahlström, Czernik, & Li, 1992; Droz et al., 1973; Di Giamberardino
et al., 1973; Goldman, Kim, & Schwartz, 1976; Lorenz & Willard, 1978).
The evidence for cytological correlates of slow axonal transport based on the
pulse-chase approach is much more limited. The principal proteins of SCa were
tubulin and neurofilament proteins, the subunits of microtubules and neurofilaments,
respectively (Black & Lasek, 1980; Hoffman & Lasek, 1975). Thus, it was hypoth-
esized that SCa represented the transport of these cytoskeletal polymers. Based on
the close similarity in transport kinetics of tubulin and neurofilament proteins, the
initial suggestion was that microtubules and neurofilaments moved as a network
of interacting polymers. However, as subsequent work revealed subtle differences
between tubulin and neurofilament protein transport (McQuarrie, Brady, & Lasek,
1986) and structural studies indicated limited interactions between neurofilaments
and microtubules (Brown & Lasek, 1993; Price, Paggi, Lasek, & Katz, 1988), the
view of SCa evolved to the independent movement of microtubules and
neurofilaments.
FIGURE 1
Axonal transport of proteins in hypoglossal and retinal ganglion cell axons of guinea pigs.
(These data are reprinted with permission from Tytell et al. (1981).) Panel (A). The
distribution of radioactive proteins in the hypoglossal nerves of guinea pigs 3 h (upper graph)
or 15 days (lower graph) after injecting radioactive amino acids into the hypoglossal nucleus
1. Pulse-Labeling studies of axonal transport 5
The only additional evidence to support the hypothesis that neurofilaments

moved in SCa was that neurofilament proteins were quantitatively assembled into
neurofilaments in axons (Black, Keyser, & Sobel, 1986; Morris & Lasek, 1982).
However, a small fraction of unassembled proteins could reasonably go undetected,
thus limiting the power of this observation. If tubulin is transported in the form of
microtubules, then microtubule-associated proteins should be cotransported with
tubulin. In this regard, minor proteins move with tubulin in SCa that have mobilities
similar to tau (Black & Lasek, 1980), a major axonal microtubule-associated pro-
tein. While an early study suggested that these may be tau (Tytell, Brady, & Lasek,
1984), subsequent analyses using two-dimensional gel electrophoresis indicated that
they are chartins (Oblinger & Black, unpublished data), a family of microtubule-
associated proteins distinct from tau. Thus, at least one microtubule-associated pro-
tein is cotransported with tubulin. However, other axonal microtubule-associated
proteins move faster than tubulin at rates in the range of SCb (Ma, Himes, Shea,
=
(the location of the neuron cell bodies whose axons form the hypoglossal nerve). Distance is
from the hypoglossal nucleus. At 3 h after injection, a well-defined wave which corresponds
to the FC is apparent, while at 15 days, two waves are apparent which correspond to SCa and
SCb. Panel (B). Comparison of the proteins comprising SCa, SCb, and FC of retinal ganglion
cell axons of guinea pigs using one-dimensional polyacrylamide gel electrophoresis.
Segments of the optic nerve and tract, which contain the retinal ganglion cell axons, were
obtained at 6 h, 6 days, or 38 days for proteins of FC, SCb, or SCa, respectively. FC and SCb
each consists of many polypeptides, whereas only five polypeptides account for the majority
of material transport in SCa. Even by one-dimensional gel electrophoresis, it is apparent that
any of the transported proteins appear in only one transport component (see the bands
highlighted by brackets). Note: the radioactive bands below tubulin in the SCa profile are not
transported in SCa but represent trailing proteins of SCb. Known polypeptides are indicated:
C ¼ clathrin, A ¼ actin, NFL, NFM, NFH ¼ low, middle, and heavy neurofilament subunits,
TUB ¼ tubulin. Apparent molecular weight is indicated on the left. Panel (C). Comparison of
the proteins comprising SCa, SCb, and FC of retinal ganglion cell axons of the guinea pig
using two-dimensional isoelectric focusingdpolyacrylamide gel electrophoresis. The
approximate pH gradient of each gel is indicated on the bottom and apparent molecule
weight is indicated on the left. This high-resolution technique shows that with very few
exceptions, each transported protein is present in only one rate component. The one
exception is the protein spot highlighted with parentheses in the samples of SCa and SCb.
Another protein present in more than one rate component is tubulin, which in peripheral
motor and sensory neurons, is transported in SCa and SCb; however, in retinal ganglion cell
axons, tubulin is only in SCa. Proteins of known identity when these data were originally
published are identified in the figures and include neurofilament subunits (NFH, NFM, NFL)
and tubulin (TUB), nerve-specific enolase (NSE), creatine phosphokinase (CPK), and actin
(A). Note: clathrin heavy chain is not identified because it forms a streak that is too faint to be
seen. The smearing of spots in the gel of FC is typical and is apparently due to the
carbohydrate and lipid modifications common to FC proteins. FC, fast component; SCa, slow
component a; SCb, slow component b.
& Fischer, 2000; Mercken, Fischer, Kosik, & Nixon, 1995). Interpretation of these
data is not straightforward. First, tau, MAP1a, and MAP1b have multiple interacting
partners in addition to tubulin, some of which (e.g., actin) move in SCb, and these
interactions can be expected to impact their movement in axons. Second, live-cell
imaging suggests that tau is cotransported with tubulin (Konzack, Thies, Marx,
Mandelkow, & Mandelkow, 2007). However, when tau dissociates from microtu-
bules, it diffuses quite rapidly, faster than the average rate of tubulin transport.
Thus, the population of tau moves faster than tubulin. While the pulse-chase studies
on transport of microtubule-associated proteins provide insights into the interactions
between tubulin and microtubule-associated proteins in axons, they do not effec-
tively address their transport form.
The structural correlates of SCb are unknown. This is in part due to its compo-
sitional complexity. Hundreds of diverse proteins move in SCb which include pro-
teins of the actin and membrane cytoskeletons, enzymes of intermediary
metabolism, proteins involved in membrane trafficking, and proteins that interact
with synaptic vesicles. Actin was one of the first proteins identified in SCb (Black
& Lasek, 1979; Willard, Wiseman, Levine, & Skene, 1979). It was suggested that
actin filaments form a scaffold to which other SCb proteins bind and the resulting
complex represents an SCb cargo. However, no direct data have been published to
support this possibility.
An early insight into SCb derived from the observations that SCb proteins move
together in a vectorial manner in axons and that they are also soluble components of
axoplasm. Such a result would be difficult to explain if the proteins were freely
diffusible. Thus, it was suggested that they existed as one or more assemblies that
were conveyed by the transport machinery (Garner & Lasek, 1982; Tytell et al.,
1981). This view is supported by cell fractionation analyses which show that
many SCb proteins behave as large multiprotein complexes (Lorenz & Willard,
1978; Scott, Das, Tang, & Roy, 2011). In addition, immunoprecipitation analyses
performed under nondenaturing conditions using antibodies specific for clathrin,
an SCb protein (Garner & Lasek, 1981), isolated a complex that included clathrin,
Hsc70, and several other minor SCb proteins (Black, Chestnut, Pleasure, & Keen,
1991). This complex may represent an SCb cargo. Finally, comparisons of the trans-
port behavior of several individual SCb proteins have revealed three distinct trans-
port profiles raising the possibility of three distinct cargoes (Garner & Lasek, 1982).
While these studies support the idea that SCb proteins form higher order assemblies
that undergo transport in axons, the identity of these complexes remains to be
discovered.
This selected review has discussed some of the history that led to the structural
hypothesis of axonal transport and the initial suggestions regarding structural corre-
lates of FC, SCa, and SCb. Many of the suggestions were controversial sparking
numerous studies using pulse-chase approaches that greatly enhanced knowledge
of axonal transport. However, these studies did not resolve the controversy because
they could not unambiguously reveal the identity of individual cargoes and the
moment-to-moment details of their movements. To move forward on these issues,
2. Live-Cell imaging of axonal transport 7
new approaches based on live-cell imaging have been developed that provide direct
visualization of the cargoes as they undergo transport in living axons. These new
methods have provided compelling support for the structural hypothesis of axonal
transport.
2. LIVE-CELL IMAGING OF AXONAL TRANSPORT

2.1 FC AND THE MOVEMENT OF VESICULAR CARGOES
Early studies using time-lapse optical imaging of living axons revealed the move-
ment of mitochondria and heterogeneous populations of roughly spherical objects
near the resolution limit of the light microscope (Forman, Padjen, & Siggins,
1977; Kirkpatrick, Bray, & Palmer, 1972). The rates of movement as well as their
sensitivity to metabolic inhibitors suggested that these were fast transport cargoes.
The introduction of video-enhanced contrast differential interference contrast mi-
croscopy revealed dramatically more movement than previously obtained because
of its ability to detect structures as small as 30 nm. Early studies on axoplasm
extruded from the squid giant axon revealed a large variety of structures moving
at rates corresponding to FC (Brady, Lasek, & Allen, 1982). Subsequent studies
using correlative electron microscopy identified many of the specific cargoes as a
variety of membrane-bound structures, thereby confirming the view derived from
pulse-chase studies (Miller & Lasek, 1985; Schnapp, Vale, Sheetz, & Reese,
1985). They also established that anterograde cargoes differed from those moving
retrogradely, with the former including Golgi-derived vesicles and the latter
including endocytic vesicles and prelysosomal structures. The squid axoplasm sys-
tem also led to the discovery of kinesin, a microtubule motor that powers fast anter-
ograde transport (Brady, 1985; Vale, Reese, & Sheetz, 1985) as well as the existence
of a distinct motor that powered fast retrograde transport (Vale, Schnaapp, et al.,
1985), which was later identified as cytoplasmic dynein. The reader is referred to
numerous reviews on fast axonal transport and the motors that power this motility
that have appeared in the intervening years.
Two points regarding FC will be highlighted. First, its anterograde and retrograde
cargoes typically move persistently and unidirectionally, pausing infrequently dur-
ing their transit in the axon. Second, while moving, their rates approximate both
the maximum rates reported for FC using pulse-chase methods and the maximum
rates reported for kinesin and dynein motors in vitro. Thus, fast axonal transport rep-
resents a system for efficiently moving vesicular structures between the cell body
and axon tip. While much remains to be learned about regulatory mechanisms
that control fast transport, the interactions of FC cargoes with the transport motors
are relatively stable and the motors interact processively with the microtubule tracks
upon which transport occurs.
Mitochondria, membrane-bound structures abundant in axons, exhibit very
different transport behavior from typical FC cargoes. Mitochondria have much
slower average rates of transport compared to fast transport cargoes (Hollenbeck &
Saxton, 2005). Live-cell imaging reveals that mitochondria pause frequently during
their transport in the axon often remaining stationary for extended times and they
can also undergo changes in direction (Saxton & Hollenbeck, 2012). Yet, mitochon-
dria transport is powered by the same kinesin and dynein motors that translocate FC
cargoes. Thus, differences in transport rate and behavior do not necessarily indicate
fundamental differences in mechanism. It is the differences in the regulation of the
transport machinery that allow the machinery to generate such distinctive transport
behaviors (Brown, 2003; Saxton & Hollenbeck, 2012). This same theme will come
up again in the discussion of slow axonal transport.
2.2 SLOW AXONAL TRANSPORT AND THE MOVEMENT OF

CYTOSKELETAL POLYMERS
The first studies attempting to reveal microtubule and neurofilament transport spe-
cifically tested the hypothesis that these structures moved slowly and steadily
from the cell body toward the axon tip at the modal rate of SCa as revealed by
pulse-chase studies (Lim, Edson, Letourneau, & Borisy, 1990; Okabe & Hirokawa,
1990; Okabe, Miyasaka, & Hirokawa, 1993). The results failed to show such slow
steady movement and were interpreted as evidence that microtubules and neurofila-
ments were not transported. However, given that these studies failed to reveal any
movement at all including that known to occur, a more conservative interpretation
would have been that microtubules and neurofilaments do not move in a slow steady
manner. As discussed below, hints already existed from pulse-chase studies on slow
axonal transport that the cargoes did not move in this manner.
2.3 NEUROFILAMENTS ARE TRANSPORTED IN AXONS

While pulse-chase studies showed that the bulk of neurofilament proteins moved
slowly and steadily at a modal rate of z1 mm/day, the wave is quite broad, indi-
cating that some SCa cargoes move faster and some slower than this. The SCa
wave also broadens substantially over time, further indicating that SCa cargoes
move at a distribution of rates. In a particularly detailed analysis of neurofilament
protein transport, rates ranged from <0.01 mm/day to several tens of mm/day
(Lasek, Paggi, & Katz, 1993). They suggested that the broad distribution of rates re-
flected a fundamental feature of the transport mechanisms in which neurofilament
proteins moved with brief but rapid translocation steps interrupted by pauses. This
is similar to the situation for mitochondria, but with pauses accounting for a
much greater percentage of the transport behavior to account for the slow average
rate of neurofilament protein transport. Although speculative at the time, this view
presaged the findings of subsequent studies directly visualizing neurofilament pro-
tein transport in living axons.
Wang, Ho, Sun, Liem, and Brown (2000) were the first to directly visualize neu-
rofilament transport in living axons, followed shortly thereafter by Roy et al. (2000).
Both groups expressed GFP-labeled neurofilament proteins in cultured sympathetic

neurons. These neurons contain a relatively sparse neurofilament array in their
axons, and many axons have regions along their length with no neurofilaments.
By focusing on these gaps which have near zero background fluorescence, GFP-
labeled neurofilaments, initially located outside of the gaps were observed to
move into and through them. Detecting these movements required the use of imag-
ing parameters to reveal fast but intermittent transport. The neurofilament proteins
moved with generally brief bouts of relatively rapid transport (z0.5 mm/s) interrup-
ted by prolonged pauses and unexpectedly, movement was bidirectional though the
majority moved anterogradely. The moving proteins comprised linear structures of
up to several tens of microns in length suggesting that they were moving as neuro-
filaments. Direct confirmation of this was subsequently provided by using correla-
tive electron microscopy to show that the moving structures were indeed
neurofilaments (Yan & Brown, 2005). Thus, the slow anterograde transport of neuro-
filament proteins in SCa actually reflects the average of brief episodes of rapid bidi-
rectional transport of neurofilament polymers interspersed with prolonged pauses of
little to no movement.
Subsequent studies showed that neurofilament transport is microtubule depen-
dent (Francis, Roy, Brady, & Black, 2005) and uses the same motors that power
fast axonal transport, with kinesin and dynein mediating anterograde and retrograde
neurofilament transport, respectively (He, Francis, Myers, Yu, Black & Baas, 2005;
Uchida, Alami, & Brown, 2009). Thus, neurofilament movement in slow transport
does not represent a novel mechanism, but instead reflects a variation on the theme
for the transport of vesicular cargoes. Specifically, fast motors propel neurofilaments
within the axon, but the movement is not processive. Specialized regulatory mech-
anisms generate prolonged pauses in this movement, resulting in a slow rate when
averaged over time. The specifics of this regulation are the subject of active
investigation.
In the years since these studies first appeared, Brown and colleagues have
continued to dissect neurofilament transport, revealing many novel details. One
goal has been to determine whether the transport behavior of individual neurofila-
ments as observed in cultured neurons imaged over short time frames can explain
the transport behavior of neurofilament proteins in axons observed over long time
frames in vivo with the pulse-chase approach (Brown, Wang, & Jung, 2005; Li,
Jung, & Brown, 2012). To address this, they developed computational models of
neurofilament transport employing the parameters for neurofilament transport rate,
directionality, and pausing observed in their studies. One essential feature of the
model is that neurofilaments move linearly and independently within axons, mostly
in the anterograde direction, but also retrogradely. In addition, individual filaments
cycle between distinct states of active transport and pausing, such that they spend
approximately 97% of their time pausing, while the remaining time, they move at
relatively fast rates. The model recapitulates the in vivo transport kinetics with
remarkable fidelity. Thus, the essential features of neurofilament protein transport
seen with the pulse-chase approach can be fully explained by the known properties
of neurofilament polymer transport seen by live imaging of cultured neurons. Over

the years, it has been suggested that neurofilament proteins may also undergo trans-
port in a form other than as neurofilaments. While this remains a formal possibility,
the available data indicate that neurofilaments constitute the principle transport form
of neurofilament proteins.
2.4 MICROTUBULES AND SLOW AXONAL TRANSPORT

Several studies have demonstrated that microtubules can redistribute within growing
neurons from the cell body into the axon and from the axon into the growth cone
(Ahmad & Baas, 1995; Slaughter, Wang, & Black, 1997; Yu, Schwei, & Baas,
1996). Though not directly observed, it was inferred that active transport accounted
for the redistribution. With the development of methods to reveal neurofilament trans-
port, it was natural to apply them to the issue of microtubule transport. The methods
clearly revealed tubulin moving in living axons, and like neurofilaments, the tubulin-
containing cargo moved rapidly but intermittently with an average rate in the range
reported for tubulin transport as seen in the pulse-chase studies (Hasaka, Myers, &
Baas, 2004; He, Francis, Myers, Black, & Baas, 2005; Wang & Brown, 2002). The
movement was bidirectional, though mostly anterograde, and during bouts of move-
ment the rate was typical of that seen with fast motors, z1e2 mm/s, but was interrup-
ted by pauses. Strikingly, the moving structures were short, z1e5 mm in length
(average ¼ 2.7 mm), and structures typical of the length of axonal microtubules
(many tens of microns long), were not observed to move. It was thus suggested
that short microtubules are conveyed rapidly but intermittently by slow axonal trans-
port while long microtubules are stationary (Baas, Nadar, & Myers, 2006).
Several observations support the view that long microtubules are not transported
in axons. For example, Chang, Svitkina, Borisy, and Popov (1999) used speckle mi-
croscopy to reveal individual axonal microtubules in living axons, and none of these
polymers was observed to move. In another approach, microtubule plus ends were
tagged with fluorescent tip-binding proteins and then imaged to see whether the
polymers moved. It is expected that the plus ends will advance as the microtubules
elongate. If they also undergo transport, then the rate of advance will exceed that due
to microtubule elongation alone. However, in no case was this observed (Kim &
Chang, 2006; Ma, Shakiryanova, Vardya, & Popov, 2004). As these studies imaged
large numbers of microtubules, if microtubule transport occurred, even infrequently,
it should have been detected. Thus, the conclusion that such transport does not occur
is reasonable. However, this needs to be qualified as the studies did not restrict
analyses to microtubules of particular length, but examined any polymer that could
be detected. As most axonal microtubules are many tens of microns in length
(Bray & Bunge, 1981), the findings reasonably apply to such long polymers.
Whether they apply to short microtubules is unknown, and given the results by
the Brown and Baas labs discussed above, they very well may not.
A key question in the studies by the Brown and Baas labs is whether the moving
tubulin-containing structures are in fact short microtubules. Given that tubulin
assembles into microtubules, this seems reasonable. However, as this has not been
directly tested by fixing tubulin-containing structures undergoing transport and im-
aging them by electron microscopy, uncertainty remains. The movement of tubulin-
containing structures that are not microtubules has been reported (Hollenbeck &
Bray, 1987). The majority move retrogradely, are spherical to oval in shape, and
are associated with membrane-bound structures. Thus, they seem unrelated to the
filamentous tubulin-containing structures of slow transport. Ma et al. (2004) have
also observed short filamentous tubulin-containing structures move in axons, but
have argued that these are not microtubules because they differ from microtubules
in fluorescence intensity. However, this conclusion is not supported by their
own data showing a transported tubulin-containing structure that is similar in fluo-
rescence intensity to microtubules elsewhere in the same images (see their
Figure 3(A)). Finally, it has been reported that brefeldin A blocks all slow axonal
transport, including the movement of tubulin (Campenot, Soin, Blacker, Lund,
Eng, & MacInnis, 2003). Because brefeldin A disrupts the Golgi complex and pre-
vents the formation of Golgi-derived vesicles that are the cargoes of FC, it was sug-
gested that slow axonal transport materials move by transient association with fast
transport cargoes. Recent support for this idea has been obtained for some SCb pro-
teins (see below), and thus it is a formal possibility for other slow transport cargoes
such as neurofilaments and tubulin. However, in these experiments, brefeldin A
treatment blocked the transport of all cargoes, including mitochondria. As mito-
chondria transport should not be affected by brefeldin A (Tang et al., 2013), the com-
plete block of transport in the experiments by Campenot et al. (2003) raises concern
of off target effects.
At present, the only independent evidence that these short tubulin-containing
structures are microtubules derives from studies of tau (Konzack et al., 2007).
These authors expressed various tau constructs in cultured neurons and examined
tau diffusion, tau association with microtubules, and tau transport. The studies
demonstrate that tau diffuses remarkably fast in axoplasm (D z 3 mm2/s) and
that tau association with microtubules exhibits a high exchange rate (t1/2 z 4 s).
Thus, diffusion is adequate to distribute tau throughout shorter axons (z1 mm).
However, as length increases beyond this, active transport is required to ensure de-
livery of tau to the distal axon. In terms of transport, the authors hypothesized that
tau was transported in association with microtubules, and used procedures similar
to that have revealed tubulin transport to visualize tau transport. Briefly, in neurons
expressing fluorescent tau, photobleaching was used to create a gap in the fluores-
cence of tau along the axon, and then the gap was imaged to determine whether
fluorescent tau located outside of the gap moved into and through the gap.
When tau with four microtubule-binding repeats was expressed, transport of
discrete structures was not observed. Given the short residence time of tau on mi-
crotubules combined with its rapid diffusion, this is expected; the fluorescent tau
would spend too little time associated with microtubules to detect its movement.
To increase the chances of detecting tau on moving microtubules, the authors
also expressed tau engineered to contain eight repeats. The eight-repeat tau resided
significantly longer on axonal microtubules and when used in the transport assay,
2- to 6-mm long filamentous structures were seen to move into and through the
gaps. The movement occurred in both anterograde and retrograde directions and
exhibited stop-and-go characteristics with brief bouts of fast transport (0.2e
2 mm/s) interrupted by pauses. The transport of these tau-containing structures
strikingly resembles that of tubulin-containing structures. It is noteworthy that
the manipulation that led to the detection of tau transport specifically involved
increasing the number of microtubule-binding domains on tau. Thus, the most
parsimonious explanation of the data on tubulin and tau transport is that tubulin
is transported as short microtubules and tau transport reflects its association with
transported microtubules.
It has been argued that microtubule transport is important for establishing the
microtubule polarity pattern of the axon, for the expansion of the axonal microtubule
array during growth and development and its maintenance in the adult (Baas, 2002;
Baas & Ahmad, 1993; Black, 1994). Impairment of microtubule transport in axons
may also be a factor in neurodegenerative diseases by compromising the axonal
microtubule array and thereby the various transport processes that depend upon it
(Baas & Mozgova, 2012). All of these ideas are based on the assumption that micro-
tubules are transported in axons, and while a strong case for this can be made, some
uncertainty remains. It is imperative to directly test whether these tubulin-containing
structures are indeed microtubules and hopefully move past this lingering uncer-
tainty. Methods are available for doing this using reagents that both fluoresce and
can be seen using the electron microscope. While such experiments pose technical
challenges, the effort will be worth the outcome because the issue will be resolved
once and for all, and the outcome will provide essential direction for the field to
move forward.
2.5 SCb AND THE MOVEMENT OF SOLUBLE PROTEINS OF

AXOPLASM
In some respects, little progress has been made in deciphering SCb, whereas in other
respects great strides have been made. With regard to the former, to explain the
movement of the 200þ soluble proteins of SCb, it was hypothesized that they
assemble into multiprotein complexes that are the cargoes of SCb (Garner & Lasek,
1982; Lorenz & Willard, 1978). While recent studies have provided further support
for the hypothesis that SCb proteins assemble into multiprotein complexes (Scott
et al., 2011; Tang, Das, Scott, & Roy, 2012), the identity of the complexes and their
possible relationship to cytological structures of the axon remain unknown. On the
other hand, substantial progress has been made in dissecting the transport mecha-
nisms for these proteins. As described below, the theme of rapid but infrequent
movements also figures important for SCb.
Initial studies expressed GFP-tagged SCb proteins (a-synuclein, synapsin-1,
glceraldehyde-3-phosphate dehydrogenase) in cultured hippocampal neurons, and
used imaging parameters to detect rapid but intermittent movements (Roy, Winton,
Black, Trojanowski, & Lee, 2007; Roy, Winton, Black, Trojanowski, & Lee, 2008).
These studies focused on thin axons in which the GFP-tagged SCb proteins appeared
as occasional discrete puncta above a more diffuse distribution. While many puncta
remained stationary during imaging, some moved at rates comparable to FC (z1e
2 mm/s). Such movements were relatively infrequent and they were interrupted by
pauses of variable duration. Furthermore, while most puncta moved anterogradely,
some moved retrogradely. These results showed that SCb proteins can move bidirec-
tionally in a stop-and-go manner. It was argued that the overall slow rate of transport
reflected the average for the population of the time spent moving rapidly and the
time spent pausing.
The key findings of these studies not fully appreciated at the time derived from
direct comparisons of the transport behavior of SCb and FC cargoes in individual
axons. Neurons coexpressing red-tagged a-synuclein (and later synapsin-1 (Tang
et al., 2013)) and green-tagged synaptophysin, an integral membrane protein of
FC, were imaged simultaneously to reveal their transport. Synaptophysin appeared
as small puncta and exhibited typical FC behavior as reported by others, with syn-
aptophysin puncta moving frequently and highly persistent. This was in marked
contrast to the infrequent and less persistent movements of SCb puncta. However,
SCb and synaptophysin puncta exhibited nearly identical transport velocities during
bouts of movement, and furthermore, in dual imaging analyses, all moving SCb
puncta moved together with synaptophysin. This latter finding was striking and sug-
gestive of a linkage between the movement of SCb proteins and FC cargoes. Subse-
quent work by Roy and colleagues demonstrated the importance of this linkage to
the transport of at least some SCb proteins.
To further dissect the mechanisms of SCb transport, Roy and colleagues devel-
oped an assay for SCb transport in cultured neurons using photoactivatable vectors
in which bulk cargo movement and particle dynamics could be visualized with high
resolution (Scott et al., 2011; Tang et al., 2012, 2013). These studies focused on three
SCb proteins, synapsin-1a, calmodulin-dependent kinase IIa, and a-synuclein.
While these proteins have distinctive transport kinetics, I will focus on their com-
monalities. The results obtained show that the bulk of these proteins moves with
an anterograde bias at a rate of z0.01 mm/s, which is similar to the rates reported
for SCb proteins based on pulse-labeling studies. The transport requires microtu-
bules, microtubule motors, and ATP.
It has not been possible to visualize discrete movements within the bulk popula-
tion of SCb proteins presumably because individual movements are too brief to cap-
ture and/or the vectorially moving proteins do not stand out from their neighbors that
are just diffusing. However, a minor subpopulation of these SCb proteins appears as
discrete particles that exhibit intricate transport kinetics. During their movement,
transport rates are relatively fast, z1e2 mm/s, but the duration of movement is var-
iable, ranging from a few seconds to a few tens of seconds (the original live-cell im-
aging studies by Roy et al. (2007) focused on this minor subpopulation of SCb
cargoes). It is assumed that movement within the wave exhibits transport kinetics
similar to these particles, but for much shorter durations. Simulations were
developed to test specific mechanisms that could explain both the slow advance of
the bulk of SCb proteins as well as the more persistent particle movements, focusing
specifically on synapsin-1 transport. The model that best fit the data involved tran-
sient association of synapsin-1 with mobile units that moved persistently with a
range of rates typical of microtubule motors and with an anterograde bias. The as-
sociation of synapsin-1 with the mobile units occurred with a range of interaction
strengths, such that most movements were of short duration (1 s) and distance
(1 mm), although a minor fraction persisted for many seconds and moved many
microns. Given the biochemical evidence suggesting that synapsin-1 along with
other SCb proteins exist as multiprotein assemblies, this suggests a model in which
complexes of SCb proteins containing synapsin-1 transiently engage with motors,
either directly or indirectly, resulting in an overall slow anterograde advance within
the axon.
Given the dual imaging analyses showing that SCb cargoes are cotransported
with synaptophysin (Roy et al., 2007; Scott et al., 2011), the vesicular cargoes of
FC that contain synaptophysin are logical candidates for the mobile units. Direct
support for this possibility was obtained by showing that manipulations that sup-
pressed FC similarly suppressed synapsin-1 transport (Tang et al., 2013). In addition,
the transport of synapsin-1 was dependent on its domains that interact with vesicular
structures. Refinements of the simulation parameters suggest a model in which syn-
apsin-1 assembles into multiprotein complexes that have an affinity to vesicular
cargoes of fast transport. The synapsin-1 complexes and vesicles interact stochasti-
cally, with most synapsin-1 complexes interacting transiently and thus advancing
slowly within the axon, whereas a minor subset interacts for longer periods and
so moves with FC.
While the extent to which this model applies to other SCb proteins is unknown,
the finding that some of the synapsin-1 moves together with two other SCb proteins,
a-synuclein and glceraldehyde-3-phosphate dehydrogenase (Roy et al., 2007), sug-
gests some generality. However, SCb is compositionally very complex, containing
200þ different proteins, and these are likely organized into multiple cargo com-
plexes (Black et al., 1991; Garner & Lasek, 1982; Lorenz & Willard, 1978; Roy
et al., 2007). A number of SCb proteins are able to interact directly or indirectly
with membranes (for example, a-synuclein, spectrin, actin, clathrin) and so may
move via transient associations with FC cargoes in a manner resembling that of syn-
apsin-1. However, it is also possible that SCb complexes are transported directly by
molecular motors in a manner that results in an overall slow transport within the
axon. Since its initial description as a discrete component of axonal transport (Black
& Lasek, 1979; Garner & Lasek, 1982; Willard et al., 1974), SCb has been a mys-
tery. Many of its proteins still remain to be identified and the current understanding
of their organization in the axon is limited and has not advanced much beyond those
of the early pulse-chase studies. However, the work of Roy and colleagues has pro-
vided a mechanistic understanding of the transport of select SCb proteins and the
next several years promise to reveal many new insights into this still enigmatic
component of axonal transport.
References 15
3. SUMMARY
In 1980, Raymond Lasek published an article on axonal transport entitled “Axonal
Transport: A Dynamic View of Neuronal Structures” in which he emphasized the
close relationship between axonal transport and the fine structure of the axon. He
argued that the structures observed in axons by electron microscopy are the cargoes
of axonal transport. In this view, studies of the fine structure of the axon and of
axonal transport provide highly complementary information. Specifically, cytolog-
ical studies provide a snapshot in time of the organization of axonal structures,
whereas studies of axonal transport provide information on the orderly motion of
these structures over time scales ranging from seconds to days, months, and longer.
Combining this information provides a dynamic view of axonal structure. Based on
information available at that time, specific hypotheses were proposed regarding the
relationship between axonal transport and axonal structures. As new technologies
were developed that provided increasingly higher resolution information on the
motility of axonal components, it became possible to directly test these hypotheses,
and some were proven correct, though often in very different ways from what was
initially envisioned, whereas others were not. The contemporary picture of axonal
transport is very different from that of three decades ago, but the fundamental prem-
ise that axonal transport provides dynamic information on axonal structures has been
fully validated by contemporary studies. Indeed, this perspective is still at the heart
of many studies of axonal transport. In many cases, the transported structures are
well defined and the studies are aimed at more subtle issues of the regulation of
transport. In other cases, the connection between the transported cargoes and axonal
structure is still being defined. The increasing resolution with which these issues can
now be examined promises answers to many of the currently outstanding questions
and with these an increasingly sophisticated understanding of how axonal transport
contributes to the elaboration and maintenance of axonal structure and function.
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cipal source of actin for the axon. Brain Research, 171, 401e413.
Black, M. M., & Lasek, R. J. (1980). Slow components of axonal transport: two cytoskeletal
networks. Journal of Cell Biology, 86, 616e623.
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port motor. Nature, 317, 73e75.
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squid giant axon. Science, 218, 1129e1131.
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Another random document with
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former is the first of six such works published in 1837 as opus 35.
The prelude is the best part of it. Though here as elsewhere he
seems to have no new or interesting means to set the piano in
vibration, though he holds without change to close arpeggio figures
throughout, yet there is a breadth of style and a sweep which
approaches real power of utterance. The fugue is excellently put
together. The theme itself recalls Bach, for whom, be it mentioned,
Mendelssohn had profound and constant admiration, and whose
works his untiring labor resurrected and brought to public
performance. Still it need hardly be added that this fugue is a work of
art, more than of expression. The inversion of the theme is clever,
and there is a certain pompous grandeur in the sound of the chorale
just before the end. The other preludes and fugues in the set are
relatively uninteresting.
The Variations are worthy of study and are by no means lacking in

musical value. The theme itself was happily chosen. There is a
respectable sadness and melancholy in it far more dignified and
genuine than the sentimentalities of the ‘Songs without Words.’ The
harmonies which underlie it are hardly bold enough to dash beyond
the diminished seventh; but a number of chromatic passing notes
give the whole something like poignancy and considerable warmth.
Moreover, it suggests chromatic treatment in the subsequent
variations.
The variations themselves are full of change and offer a range of

contrast of which Mendelssohn was not often master. The effect of
the series as a whole is therefore stimulating and rather brilliant.
The first variation adds a counterpoint to the theme in groups of four

sixteenths. The counterpoint in the second is of groups of six
sixteenths. The first two variations thus seem to set the piece
gradually into a free motion, which throughout the next two grows
more vigorous and more nervous. The fifth is typical of
Mendelssohnian agitation; but it serves as an excellent introduction
to the chords of the sixth and seventh. The eighth and ninth work up
to a frenzy of quick motion. Then follow two in a suppressed and
quiet style, the first a little fugue, the second a brief and exquisite
cantilena. The twelfth is the most vigorous of the lot, a movement as
near the virtuoso style as Mendelssohn ever was able to produce.
The thirteenth is interesting by reason of the contrast between the
legato melody in the left hand and the excellent staccato
counterpoint. A short adagio, rather superior to most of the songs in
a similar style, forms the fourteenth. The fifteenth is transitional, the
sixteenth and seventeenth merely lead up to the presto at the end.
The entire group presents nothing in the treatment of the piano in
advance of Weber, if, indeed, it anywhere equals him; but it is both in
quality and in style a very fine piece of pianoforte music, which can
hardly fall under the censure to which most of his music for the
instrument is open.
There are two concertos and a concert piece for piano and
orchestra. The latter owes its form and style very clearly to Weber’s
concert piece in F minor. Both the concertos are fluent and plausible
enough; the orchestra is handled with Mendelssohn’s customary
good taste and sensitiveness; but the writing for the pianoforte is
wholly commonplace and the themes themselves of little or no
distinction.
The ‘Songs without Words’ were published in six groups of six

pieces each during his life. After his death in 1847 two more sets
appeared. The influence of all these was widely felt, particularly
among composers of mediocre gifts. Chopin had no liking for them.
In fact, Mendelssohn’s music was more than ordinarily distasteful to
him; and he is said to have declared that Mendelssohn never wrote
anything better than the first song without words. In some respects
this is true. Schumann had a great admiration for Mendelssohn;
admired his orderly style and manner. But Schumann’s individuality
was far too pronounced, especially in pianoforte predilections, to
submit to the milky sway of Mendelssohn.
In pianoforte music, William Sterndale Bennett (1816-1875) carried

on the Mendelssohn tradition quite undefiled. Bennett was more than
a pupil of Mendelssohn; he was a devoted and unqualified admirer.
His own pianoforte works are numerous, but they have suffered
something of the same malice of Fate that still preserves the ‘Songs
without Words’ chiefly for fun. They include four concertos, and
many short pieces, studies, diversions, impromptus. They have the
merits of their prototypes, clear, faultless writing and melodiousness.
A contemporary of Mendelssohn whose life led him finally to

Petrograd, is still remembered by one or two of his studies. This is
Adolf Henselt (b. 1814-89). Henselt’s work is really independent of
Mendelssohn. His style was founded upon a close acquaintance with
Weber’s. In 1836 he gave private recitals in Berlin and was
especially prized for his playing of the Weber sonatas. Two sets of
concert studies were published as opus 2; and in them is the still
famous and delightful Si oiseau j’étais. Besides these he composed
numbers of Rhapsodies, Ballades, and other short pieces in the
romantic style; all of which together show distinctly more originality in
the treatment of the piano than Mendelssohn showed.
II
Meanwhile Robert Schumann was composing sets of pieces which
have been and long will be regarded as one of the most precious
contributions of the Romantic movement to pianoforte literature.
Schumann was an enthusiast and an innovator. He was a poet and a
warm-hearted critic. He was the champion of the new and the fresh,
of self-expression and noble sentiment. In his early manhood a
strained finger resulted from over-enthusiastic and unwise efforts to
make his hand limber, and cut short his career as a concert pianist,
for which he had given up his study of the law, not without some
opposition. He turned, therefore, with all fervor to composing music
for the pianoforte, and before his long-delayed marriage with Clara
Wieck, daughter of his teacher, had published the sets of pieces on
which a great part of his fame now rests.
Schumann was steeped in romantic literature, particularly in the

works of Jean Paul Richter and E. T. A. Hoffmann; and most of his
works show the influence of these favorite writers upon him. One
finds symbolical sequences of notes, acrostics in music, expressions
of double and even triple personalities; but these things are of minor
importance in his music. The music itself is remarkably warm and
poetic, remarkably sincere and vigorous whatever the inspiration
may have been. It is happily sufficiently beautiful in itself without
explanation of the cryptograms which oftener than not lie underneath
it.
He was, as we have said, an explorer and an innovator by nature;

and his music is full of signs of it. Though his treatment of the piano
lacks the unfailing and unique instinct of Chopin, nevertheless his
compositions opened up a new field of effects. Not all of these are
successful. Experiments with overtones such as one finds, for
instance, at the end of the Paganini piece in the Carnaval can hardly
be said to be worth while. The result is too palpably an isolated effect
and nothing more. It is too self-conscious. But he was of great
significance in expanding the sonority of the instrument, in the use of
the pedal, in the blending of harmonies, in several finer touches of
technique. The combination of two distinct themes in the last
movement of the Papillons, the fluent and sonorous use of double
notes in the Toccata, the wide skips in the 'Arlequin’ and the
'Paganini’ numbers of the Carnaval, the latter with its cross-accents;
the Reconnaissance in the same series, with its repeated notes; the
rolling figures in the first movement of the Kreisleriana; these, among
other signs of his originality, are new in pianoforte music.
His compositions demand from the pianist an unlimited and a

powerful technique, yet it cannot be said of any that it is virtuoso
music. He employed his skill not so much to display as to express
his ideas. Nowhere does the pianoforte seem more the instrument of
intimate and highly romantic sentiment. Of figure work and
ornamentation there is very little. His music is not at all dazzling.
Much of it is veiled. At the most he is boisterous, as in parts of the
Faschingsschwank and the last movements of the Études
Symphoniques. He rather avoids the high, brilliant registers of the
keyboard, stays nearly constantly in the middle of things, deals in
solid stuff, not tracery.
Perhaps the most distinctive feature of his style is his frequent use of
syncopated rhythms. This becomes at times an obsession with him;
and there are many passages in his music so continuously off the
beat, that the original measure is quite lost, and the syncopation is to
all practical purpose without effect. In such passages it seems hardly
possible that Schumann intended the original beat to be kept in mind
by the accentuation of notes that are of secondary importance;
unless, of course, the interest of the music is chiefly rhythmical. Yet
in some passages of purely melodic significance this may be done
without awkwardness, producing an effect of dissociation of melody
and harmony which may be what Schumann heard in his mind.
These are problems for the pianist, but a few of them may be
suggested here. The last movement of the very beautiful concerto is
in 3/4 time. There is no change of time signature for the second
theme. This, as first announced by the orchestra in E major and later
taken up by the piano solo in B major, is none the less in 3/2 time.
Such must be the effect of it, because the passage is long and
distinct enough to force the 3/4 beat out of the mind, since no note
falls in such a way as to accent it. But when the orchestra takes up
this theme, again in E major, the piano contributes a steadily flowing
stream of counterpoint. In this it is possible to bring out the original
measure beat, throwing the whole piano part into a rhythm counter to
the rhythm of the orchestra. Such an accentuation is likewise out of
line with the natural flow of the counterpoint; yet it may be what
Schumann desired here, as well as in the following section, where,
though the orchestra is playing in 3/2 time, the pianist may go
against the natural line of his own part and bring out a measure of
three-quarter notes.
The middle section of the second movement of the great Fantasy in

C major presents the same problem. Here we have a melody in long
phrases. The notes of it are off the beat, the chords which furnish its
harmony are on the beat. Every eight measures the natural rhythm
asserts itself; yet even these periodic reminiscences of the measure
cannot serve to throw the whole melody into syncopation. The
melody is too strong and its phrases too long. More than the
occasional measures, it must, if allowed fully to sing, determine the
rhythm of the passage. So it is usually played; so, without special
effort to the contrary, it will impress the ear. Now is it possible that
Schumann intended the accompanying chords to be distinctly
accented? Such an accent, delicately applied, with the skillful use of
the pedal, will create a wholly new effect, which can be drawn from
all the succeeding passages as well.
Other passages offer no alternative. There is no way to suggest the

original beat except by movement of the body, or by grunting; both of
which are properly discountenanced. Examples may be found in the
first movement of the Faschingsschwank and elsewhere.
Most of Schumann’s pianoforte music is made up of short pieces.

Such are the Papillons, the Carnaval, the Davidsbündler Dances, the
Faschingsschwank, the ‘Symphonic Studies,’ and the Kreisleriana.
Each of these is a cycle of pieces, and is at best only loosely held
together by one device or another. The Papillons are scenes at a
fancy dress ball. The return of the first piece at the end gives a
definite boundary, as it were, to the whole. The Faschingsschwank
are pictures of a fête in Vienna. There is no structural unity to the
work as a whole. The fanciful idea upon which it rests alone holds
the pieces loosely together.
The Carnaval, likewise a scene at a fair, representations in music of

various people, sights, and sounds, is built on three series of notes
which Schumann called ‘Sphinxes’ and which he had published with
the music. It is very doubtful whether the employment of these
sequences in one form or another gives to the whole series an
organic interdependence. Only with care can the student himself
trace them, in such varied guises do they appear; and to be left in
entire ignorance of them would hardly interfere in the least with an
emotional appreciation of the music. The return at the end of some
of the movements and passages heard at the beginning, however,
rounds off the work and makes an impression of proportions.
Moreover, within the work many of the pieces lead without pause into
the next, or are without an end at all, like the Florestan, which is left
fulminating in the air.
In the Davidsbündler there is again the return at the end of familiar

phrases, but the Kreisleriana is like the Faschingsschwank without
structural unity. Yet perhaps none of the Schumann cycles is less
friable than the Kreisleriana. It is long and it is varied; but here,
perhaps more than in any other similar works of the composer, there
is a continuous excellence of workmanship and intensity of
expression.
Besides these cycles there are sets of short pieces which are
independent of each other. Such are the ‘Fantasy Pieces,’ the
Novelettes, the ‘Romances,’ and the Bunte Blätter, among others.
These may be fairly compared with the ‘Songs without Words’ of
Mendelssohn. How utterly different they prove to be, how virile and
how genuinely romantic! They are not only the work of a creative
genius of the highest order, they show an ever venturesome spirit at
work on the keyboard. Take, for example, the ‘Fantasy Pieces.’ The
first, called Des Abends, is as properly a song as any of
Mendelssohn’s short pieces which are so designated. The very
melody is inspired and new, rising and falling in the long smooth
phrases which are the gift of the great artist, not the mere music-
maker. The accompaniment appears simple enough; but the wide
spacing, the interlocking of the hands, above all, its rhythm, which is
not the rhythm of the melody, these are all signs of fresh life in
music. The interweaving of answering phrases of the melody in the
accompaniment figures, the contrast of registers, the exquisite points
of harmonic color which the accompaniment touches in the short
coda, these are signs of the great artist. It is remarkable how little
Mendelssohn’s skill prompted him to such beautiful involutions; how,
master as he was of the technique of sound, he could amble for ever
in the commonplace. And Schumann, with far less grasp of the
science, could venture far, far beyond him.
The second of the ‘Fantasy Pieces,’ Aufschwung, calls imperiously
upon the great resources of the pianoforte. There is power and
breadth of style, passion and fancy at work. It is a wholly different
and greater art than Mendelssohn’s. It is effective, it speaks, it
proclaims with the voice of genius. And in the little Warum? which
follows it, skill is used for expression. There is perhaps more
appreciation of the pianoforte in this piece, which by nature is not
pianistic, than there is in all the ‘Songs without Words,’ an
appreciation of the contrasting qualities of high and low sounds, of
the entwining of two melodies, of the suggestive possibilities of
harmony.
Take them piece by piece, the Grillen with its brusque rhythms, its
syncopations, its rapidly changing moods; the In der Nacht, with its
agitated accompaniment, its broken melodies, and the soaring
melody of the middle section, not to mention the brief canonic
passages which lead from this section back to the wild first mood;
the delicate Fabel, the Traumes Wirren with its fantastic, restless,
vaporish figures and the strange, hushed, shadows of the middle
section; and the Ende vom Lied, so full for the most part of good
humor and at the end so soft and mysteriously sad; these are all
visions, all prophecies, all treasure brought back from strange and
distant beautiful lands in which a fervid imagination has been
wandering. Into such a land as this Mendelssohn never ventured,
never even glanced. For Schumann it was all but more real than the
earth upon which he trod, such was the force of his imagination.
The imagination is nowhere more finely used than in the short pieces
called the Kinderscenen. Each of these pieces gives proof of
Schumann’s power to become a part, as it were, of the essence of
things, to make himself the thing he thought or even the thing he
saw. They are not picture music, nor wholly program music. They are
more a music of the imagination than of fact. Schumann has himself
become a child in spirit and has expressed in music something of the
unbound rapture of the child’s mind. So, even in a little piece like the
‘Rocking Horse,’ we have less the picture of the ‘galumphing’
wooden beast, than the ecstasy of the child astride it. In the Curiose
Geschichte there is less of a story than of the reaction of the child
who hears it. In the Bittendes Kind and the Fürchtenmachen this
quality of imagination shows itself with almost unparalleled intensity.
The latter is not the agency of fear, it is the fear itself, suspense,
breathless agitation. The former does not beg a piece of cake; it is
the anguished mood of desire. Only in the last two pieces does
Schumann dissociate himself from the moods which he has been
expressing. The former, if it is not the picture of the child falling
asleep, is the process itself; the latter is, as it were, the poet’s
benediction, tender and heartfelt.
The whole set presents an epitome of that imagination which gave to

Schumann’s music its peculiar, intimate, and absorbing charm. His
might well be considered the most subjective of all pianoforte music.
It is for that reason dull to practice. The separate notes of which it is
composed give little objective satisfaction. The labor of mastering
them routs utterly in most cases the spirit which inspired them. Fine
as the craftsman’s skill may prove to be in many of the pieces, it is
peculiarly without significance, without vitality, until the whole is set in
motion, or set afire by the imagination.
The most imaginative and the most fantastic of the works as a whole
is the series of twenty short pieces which make up the Carnaval,
opus 9. Here there is a kaleidoscopic mixture of pictures, characters,
moods, ideas, and personalities; the blazonry of spectacle, the noise
and tumult, the quiet absorption that may come over one in the midst
of such animation, the cool shadows beyond the edge of it wherein
lovers may wander and converse; strange flashes of thought,
sudden darting figures, apparitions and reminiscences. All is
presented with unrelaxing intensity. One cannot pick out a piece from
the twenty which does not show Schumann’s imagination at fever
heat. There is a wealth of symbolism; the Sphinxes, mysterious
sequences of notes that are common to all the pieces, and dancing
letters which spell the birthplace of one of Schumann’s early loves.
As to the Sphinxes it may be said, as before, that the coherence

which they may add can hardly exist outside the mind of the player,
or of the student who has made himself thoroughly familiar with the
work. The average listener may hear the whole work a hundred
times, learn to know it and to love it, without ever realizing that the
first intervals of the Arlequin, the Florestan, of the Papillons and
others are the same; those of the Chiarina, the Reconnaissances,
and the Aveux likewise, note for note identical. Such hidden
relationships in music are vaguely felt if felt at all. Just as two words
spelled the same may have different meanings, so may two musical
phrases made up of the same intervals be radically different in effect.
The Carnaval opens with a magnificent prelude. The first section of it

suggests trumpeters and banners, the splendid announcement and
regalia of a great fête. After this we are plunged at once into the
whirr of merry-making. Schumann’s cross-accents and syncopations
create a fine confusion; there is hurly-burly and din, a press of
figures, measures of dance, light and tripping, an ever-onward rush,
animato, vivo, presto! There is a splendid effect in the last section,
the presto. The measure beat is highly syncopated. It will be
observed that in the first eight measures the first notes of every other
measure, which are in all dance music the strongest, are single
notes. These alone keep up a semblance of order in the rhythm. By
the extension of one measure to four beats, the sequence of notes is
so changed that in the repetition of this first phrase the strong accent
falls upon a full chord, thus greatly re-enforcing the intended
crescendo.
The next two numbers in the scene are pictures of two figures
common to nearly every fair, the Pierrot and the Harlequin. The
distinction between them is exquisite. In Pierrot we have the clown,
now mock-mournful and pathetic, only to change in a second and
startle with some abrupt antic. Harlequin, on the other hand, is
nimble and quick, full of hops and leaps. At the end of the Pierrot, by
the way, there is the chance to experiment with the pedal in
overtones. The sharp fortissimo dominant seventh, just before the
end, will set the notes of the following chord, all but the fundamental
E-flat, in vibration if the pedal is pressed down; so that the keys of
this second chord need hardly to be struck but only to be pressed.
And when the pedal is lifted, this second chord will be left still
sounding, by reason of the sympathetic vibration which was set
about in its strings by the loud chord preceding.
Pierrot and Arlequin are professional functionaries at the fair. We are

next introduced to a few of the visitors. There is a Valse noble and
then Eusebius. Schumann imagined within himself at least three
distinct personalities of which two often play a rôle in his music. One
is active and assertive. He is Florestan. The other is Eusebius,
reflective and dreamy. Here, then, is Eusebius at the fair, wrapped
about in a mantle of gentle musing. His page of music in the
Carnaval is one of the loveliest Schumann ever wrote. Elsewhere,
too, the contemplative young fellow speaks always in gentlest and
most appealing tones; as in the second, seventh, and fourteenth of
the Davidsbündler Dances, all three of which are subscribed with a
letter E.
In the Carnaval, as in the Dances, Florestan breaks roughly into the

meditations of Eusebius. He works himself into a very whirlwind of
energy; and then Schumann, by a delicious sense of humor, lets the
artful Coquette slide into his eye and put an end to his vociferations.
To her there is no reply but the gentle, short Replique. Are the
Papillons which follow masqueraders? The horn figures of the
accompaniment bring in a new group to the fair, fresh from the outer
world. They are gone in a flash, and their place is taken by three
dancing letters, 'As,’ C, and H; As being German for A-flat, and H for
B. And these letters spell the birthplace, as we have said, of one of
Schumann’s early loves.
The love of his whole life follows—Chiarina, his beloved Clara; and,
as if with her were associated the loveliest and most poetic of
pianoforte music, he calls Chopin to mind. Chopin at this fair! It is a
fantastic touch. More than when Eusebius speaks, the background
of gay dancers and masqueraders fades from sight. For a moment
Chopin is in our midst. Then he has vanished. And at once another
thought of Clara, this time as Estrella; then an acquaintance in the
throng. He has seen a face he knew, it is a friend. It is the Sphinx of
Chiarina in the music. Is it she he recognized? Are the lovely
interchanges in the middle section conversations with her? If so,
their mood is light. They have met at a fair. They are in the merry-
making.
Two more professionals, masquers this time—the world-favorites,

Pantalon and Colombine; and at the end of their piece an exquisite
thought of Schumann’s. Then the German waltz, simplicity itself; and
in the midst of it none other than the wizard, Paganini! Surely, there
was never a stranger trick of thought than that which thus placed
Paganini in the midst of a simple, tender German waltz. He vanishes
in a puff of smoke, as conjured devils are supposed to do; and the
waltz goes on, as if all this intermission had been but a flash in the
air above the heads of dancers too absorbed in their pastime to note
such infernal phenomena.
After the waltz, a lover’s confession, hesitating but enraptured; and

then a Promenade. There is full feeling, there is delight and ecstasy.
Our lover whirls his maiden from the fair. Farther and farther they go,
hand in hand, into the shadowy, calm night. Fainter and fainter the
sounds of revelry, till all is silence.
There is a pause. The lovers are dispatched. Away with dreaming,

away with sentiment! Back into the hurly-burly and the din. Here
comes the band of David down the plaisance, hats in air, banners
flying, loudly cheering. These are the sons of the new music. These
are the champions of the new era of freedom, these the singers of
young blood. More and more reckless, madder and more gay!
Spread consternation abroad among the Philistines, put the learned
doctors to rout, send them flying with their stale old tunes and laws!
So the Carnaval ends, with the flight of the old and dusty, and the
triumph of the enthusiasm of youth.
Here is a phantasmagoria unmatched elsewhere in music. It is very

long. It is too long; and, judged as a whole, the work suffers in
consequence. It is overcrowded with figures, too full of symbolism;
and the ear tires, the attention wearies. Yet there is not a piece in it
which one would be willing to discard. All are beautiful and new and
full of life. Many present something peculiar to Schumann, the fruit of
his imagination, which is in advance of most of the music of his time.
It must occupy an important place in the history of pianoforte music,
as representing one of the finest accomplishments directly due to the
influence of the Romantic movement.
III
The other cycles of Schumann comparable to it are the Papillons,
opus 2, the Davidsbündler Tänze, opus 6, and the
Faschingsschwank aus Wien, opus 26. The first of these is short and
slight, but of singularly faultless workmanship and rare charm. The
last must be cherished for the Romanza, the Scherzo, and the
splendid Intermezzo; but the first movement is rather out of
proportion, and parts of the last are perfunctory and uninteresting.
Most of the Dances of the Davidsbündler are beautiful. The series is,
however, much too long and too loose to be regarded as a whole.
There are passages of unsuccessful workmanship, notably in the
third; some of the dances are rambling, some rather commonplace.
On the other hand, many may be ranked among the best of
Schumann’s compositions. The second, seventh, and fourteenth
have been mentioned as among the beautiful utterances of
Eusebius; the fifth is less distinguished but is delightful pianoforte
music. Florestan does not make quite such a good impression,
except possibly in the fourth and the twelfth. The fifteenth speaks for
both Florestan and Eusebius; and the E-flat major section is
splendidly rich and full-throated music. The last dance of all is like a
happy, wayward elf waltzing along in the wake of more substantial
dancers. The series may properly end with the seventeenth; but, as
Schumann said, though Eusebius knew well that the eighteenth was
quite superfluous, yet one could see by his eyes that he was blissful
over it.[32]
Both the ‘Symphonic Studies’ and the Kreisleriana stand apart from
the works previously discussed. The former, opus 13, was written in
1834, the latter, opus 16, in 1838. A brief glance at opus 1, the
‘Abegg’ variations, written in 1830, will serve to make clear the
immense progress Schumann made in the art of composition in the
brief space of four years. The early work is by no means lacking in
interest. Schumann reveals himself in nearly every page. The theme
itself is made up of the notes a, b, e, g, g, spelling the name of the
honorable lady to whom the variations were dedicated. In the middle
of the last movement he experiments with a new style of
diminuendo, allowing a chord to die away by separate notes, till only
one note of it is left sounding. He tried the same effect again at the
end of the Papillons. But the workmanship, though clever, is for the
most part conventional. The statement of the theme is laughably
simple, particularly the ‘echoes,’ pianissimo, in broken octaves. Such
a device recalls the ‘Maiden’s Prayer’ and fountain curls. The
variations show a fine ear for pianoforte effects. The first especially is
in virtuoso style and makes more use of the upper registers of the
keyboard than is common in the later works. But the harmonies,
though richly altered, are conventional, and so are the figures. The
third, fourth, and fifth might have been written by Hummel.
The ‘Symphonic Études’ are immeasurably broader and more

original. They are written as variations; but Schumann confines
himself very little to the conventional scheme; and the third and ninth
are not variations at all, but études made up of wholly extraneous
ideas. The theme itself is dignified and rich, and its statement in the
sonorous middle registers of the piano is impressive. In the first
measures of the first variation there is little or no suggestion of the
theme save in harmony. The opening phrase is given low down,
repeated in higher registers, till the music has climbed nearly four
octaves; at which point a phrase of the theme makes its appearance.
Toward the end of the variation the same phrase is heard again; but
the whole is distinctly dominated by the figure announced in the first
measure.
In the second variation the theme is carried throughout in the bass;
but a beautiful new melody is imposed upon it which carries the
burden of the music. The third of the series is unrelated to the theme
except in key. It is a study in light, wide, staccato figures for the right
hand; under which the left hand carries a suave and expressive
melody. In the next movement, the theme is treated consistently as a
canon at the octave. The next is at once a study in a capricious
dotted rhythm and a subtle variation of the theme. And in the
following, the sixth, the theme is wholly prominent in both hands, the
left anticipating the right by the fraction of a beat. The seventh is a
magnificent study for the movement of the arm from one group of
notes to another. It is in E major, and the theme makes but an
occasional and fragmentary appearance. The eighth is a study in
sharp cross-accents, the theme again wholly concealed, except for
its harmonies; the ninth a study in double notes and octaves for the
wrist. The tenth, eleventh, and twelfth are high-water marks in
Schumann’s treatment of the pianoforte, both in brilliant and poetic
effects. Particularly worthy of study are the accompaniment figure in
the latter, with its rich shimmering of harmony, and the skillful
interweaving of two melodies in the fashion not long before
employed in the short Warum?. The finale, which, with the repeats
Schumann incorporated into it, is far too long, practically exhausts
the power of the piano in big chordal effects.
There is but little trace of the composer of the Abegg variations in

these imposing and wholly beautiful studies. Schumann shows
himself in them such a master of the pianoforte as has no need to
display his wares, but may let their intrinsic richness and splendor
speak for them. Only in the last of them does he lay himself open to
the criticism of having treated the piano in a style too nearly
orchestral, which expects from the instrument a little more than it can
furnish. Elsewhere in the series the very spirit of the piano speaks, a
noble and moving language, full of imagery and of color. The obvious
virtuoso trappings of Weber are left far behind. We are on one of the
great heights of pianoforte literature.
Schumann considered the Kreisleriana to be his best work for the
piano alone. It was inspired by the character of Johannes Kreisler,
an eccentric, highly gifted kapellmeister who figured in the tales and
musical papers of E. T. A. Hoffmann.[33] Just what it means few will
venture to suggest. The last movement may recall the account of the
last appearance of Kreisler on this earth, as he was seen hopping
along the road beyond the town, with a red hat on the side of his
head and a wooden sword by his side. Dr. Oskar Bie quotes from
Hoffmann in connection with the second movement, the tale of the
young girl who was lured to a magic oak by the sound of a lute, and
there killed; whose heart grew into a twining rose-bush.[34] In the
main, however, the music eludes analysis. It is eccentric. Though full
of the mannerisms of Schumann, much of it presents an unfamiliar
mood of the composer. The moods of it are different from the moods
of the ‘Carnival,’ the ‘Symphonic Études,’ the ‘Fantasy,’ the ‘Scenes
of Childhood.’ On the whole, it lacks the warmth of his other works. It
is fantastic, and not unfrequently grotesque; parts of it strangely
deliberate. Many pages of it are out of the usual and consequently
baffling. It is more involved, too, in workmanship, and the separate
movements full of contrasts that seem to be vagaries. Schumann
has, of course, here as elsewhere put himself into the person of his
inspiration; and the result is a tribute to the power of his imagination.
Never was music more fantastic, less consequential.
It is, on the other hand, superb. The opening movement alone, with
its figures like short waves in a windy sea, its sharp cross-accents,
its filmy, elusive trio, is a masterpiece. The second movement is
unbalanced, yet at times most wondrously beautiful. The opening
theme in itself is inspired, though it is perhaps overworked. But what
is the meaning of the harsh chords which interrupt it and shatter the
mood which it might else instill? The style is polyphonic in places;
there are inner melodies that slide long distances up and down the
keyboard, oftenest in tenths. The two intermezzi furnish a welcome
contrast to the intense subjectivity of most of this second movement.
After the second there comes one of the loveliest pages in all
Schumann’s pianoforte music.
The third movement is built on a restless, jerky figure, in ceaseless
movement. There are strong accents and unusual harmonies. A
middle section offers yet another happy instance of Schumann’s skill
in dialogue between two melodies, such as we have already noticed
in Warum? and the eleventh of the ‘Symphonic Études.’ The
movement is somewhat slower than the main body of the piece, but
a strange sort of half-accompaniment does not allow the
restlessness to subside altogether.
The fourth and sixth movements are slow. In both there is some
thickness of scoring, a sinking too deep into the lower registers. Both
are about the same length and both are constructed on the same
plan; consisting of an incompleted, or broken, melody of the most
intimately expressive character, a few measures of recitative, the
melodious phrases again—in the one wandering down alone into the
bass, disappearing rather than ending, in the other not completing
itself, but developing into a contrasting section. In both there are
these contrasting sections of more articulate and more animated
music; and in both there is a return of the opening melody. There is
wonderful music in these two short movements; but it is mysterious,
fragmentary and incomplete, visionary, as it were, and without
definite line.
The remaining movements escape language. The fifth is full of

changing moods; the seventh more than the others, consistent, this
time in a vein of something like fury. The eighth and last is delicate
and whimsical. The right hand keeps to a light, hopping figure most
of the time; the left hand has little more than long single notes, which
pursue a course of their own, without regular rhythm.
There is a lack of titles, there is no motto, there is even no mark of

Florestan and Eusebius. This most whimsical, most subjective, and,
in many ways, most beautiful and most complicated of Schumann’s
creations, stands before us, then, with no clue to its meaning except
its title. This, as we have said, refers us to a half-crazy, fantastical
musician. There is more in the music than lunacy, full of vagaries as
it is. There is much poetry, a clearness and sanity in diction,
inconsequential as the thought may be, a mastery of the science of
music. Yet it is not surprising if some, bearing in mind the
preternatural activity of Schumann’s imagination even in early
manhood, and the breaking-down of his mind toward the end of his
life, will hear in this music a note of something more tragic than
whimsical fancies, will feel that Schumann has strayed perilously far
afield from the world of orderly nature and warm blood.
A few short pieces that Schumann published, like the Novelletten,

are not held together in a cycle. In these the humor is prevailingly
happy and active, the workmanship clear, and the form well-
balanced. Fine as they are, in listening to them separately one
misses something of Schumann. The man was a dreamer. He sank
himself deep into moods. He lived in complete worlds, created by his
fancy. A single piece like one of the ‘Novelettes’ hardly initiates the
listener into these wide domains. Fully to put ourselves in touch with
Schumann we must wander with him, and in the course of our
wandering, drift farther and farther into his land of phantoms.
Four works in broad form must be reckoned among his greatest

compositions. These are two sonatas: one in F-sharp minor, opus
11, one in G minor, opus 22; the great ‘Fantasy’ in C major, opus 17;
and the concerto for pianoforte and orchestra in A minor, opus 54. It
is hard to estimate the worth of the sonatas. That in F-sharp minor is
rambling in structure, and too long; yet there are pages of splendid
music in it. The introduction is full of a noble passion and strength;
the first theme of the first movement has a vitality which, better
ordered, would have made of the whole movement a great
masterpiece; and the second theme is undeniably beautiful. But
transitional sections and the development are monotonous and too
little restrained. The second movement, making fuller use of the
themes hinted at in the introduction, is wholly satisfying; the scherzo,
likewise, with its grotesque Intermezzo and mock-heroic recitatives.
But in the last movement again there is far too much music, far too
little art; and, despite the healthy vigor of the chief theme, the piece
staggers rather than walks.
The sonata in G minor is more concise, is, indeed, perfect and clear-
cut in form. All of it is lovely, particularly so the Andantino and the
Rondo. There is perhaps too much restlessness in the first
movement and, consequently, too little variety. It is all flame and no
embers.
The Fantasy is colossal. It is said that Schumann intended the first

movement to represent ruins, the second a triumphal arch, the last a
starry crown. Subsequently he changed his intention; but something
of these original characteristics still remains. The first movement is a
strange mixture of stark power, tenderness, and romantic legend. It
is not hard to find in it the groundwork of the triplex form. There is a
first theme, the dominant theme of the movement, strangely gaunt
and bare; and a contrasting theme beautifully melodious which
Schumann associated with his beloved Clara. These two themes are
presented fairly regularly in the first section of the movement; and
the last section brings them back again, as in the triplex form. But
there is a broad middle section, in legendary character, which
presents a wealth of different material, some of which has been
freely used between the first and second themes in the first section.
The whole is greatly expanded, full of pauses, passages of
unrestrained modulation. The effect is truly magnificent.
The second movement exceeds the finale of the ‘Symphonic Études’

in triumphant vigor. The last movement is long, richly scored, exalted
in sentiment. The endings of the three movements, especially of the
first and last, are inspired, wholly without trace of the commonplace.
It is one of the truly big works for the piano, lacking perhaps in
subtlety and refinement of technique, sometimes a little awkward
and out of proportion, but full of such a richness of harmony and
melody, of such passion, strength, and romance, of such poetry and
inspiration, as to defy criticism. It is, as we have said, colossal.
The concerto stands as a flawless masterpiece. The themes are

inspired. There is no trace of sentimentality or morbidness. The form
is ruled by an unerring and fine sense of proportion and line. It is
neither too long nor too short. There is no awkwardness, no
tentativeness, no striving for effect. No note is unwisely placed. The
treatment of both pianoforte and orchestra leaves nothing to be
desired, either when the one is set against the other or when both
are intimately blended. Though it in no way suggests the virtuoso, it
is perfectly suited to the piano, bringing out unfailingly the very best
the instrument is capable of. Thus it stands unique among
Schumann’s compositions. There must be many to whom it stands
for an ideal realized. To them it will be unique among concertos, the
most excellent, the perfect type.
With this masterpiece we may take leave of Robert Schumann, for

whom most pianists will ever have a special love. The first
movement was composed in 1841, the Intermezzo and Finale in
1845, all after his marriage in the fall of 1840. After this happy
termination to long and troubled years, his attention turned to other
branches of music, to songs, to oratorios and symphonies; and,
though he never forsook the piano entirely, the best of his work for it,
with few exceptions, was left behind him. The ten years between
1830 and 1840 saw its creation. In this relatively brief period all the
works we have mentioned, except the concerto, were composed.
They were the flower of his early manhood, and they bear witness in
every page to the romantic eagerness and fire of youth. In many a
measure they show a lack of skill, an excess of zeal, an over-
reaching that is awkward; but what are these in the fire of his poetic
imagination? The spirit of Schumann rises far, far above them, one
of the most ardent, soaring spirits that ever sought expression in
music. It was destined to fall back, ruined, charred, and blackened
by its own fire; but happily we have left to us in pianoforte music its
song at the height of its flight.
IV
The only worthy successor to Schumann in the realm of German
pianoforte music is Johannes Brahms. Into the hands of Brahms
Schumann may be said to have given over the standard which he

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The neuronal cytoskeleton, motor

proteins, and organelle trafficking in the

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

Academic Press is an imprint of Elsevier

For information on all Academic Press publications

proteins with quantum dot- or fluorochrome-labeled ligands or antibodies; and

Axonal transport: The

Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.06.001 1

1. PULSE-LABELING STUDIES OF AXONAL TRANSPORT

The only additional evidence to support the hypothesis that neurofilaments

2. LIVE-CELL IMAGING OF AXONAL TRANSPORT

2.2 SLOW AXONAL TRANSPORT AND THE MOVEMENT OF

2.3 NEUROFILAMENTS ARE TRANSPORTED IN AXONS

Both groups expressed GFP-labeled neurofilament proteins in cultured sympathetic

of neurofilament polymer transport seen by live imaging of cultured neurons. Over

2.4 MICROTUBULES AND SLOW AXONAL TRANSPORT

2.5 SCb AND THE MOVEMENT OF SOLUBLE PROTEINS OF

The Variations are worthy of study and are by no means lacking in

The variations themselves are full of change and offer a range of

The first variation adds a counterpoint to the theme in groups of four

The ‘Songs without Words’ were published in six groups of six

In pianoforte music, William Sterndale Bennett (1816-1875) carried

A contemporary of Mendelssohn whose life led him finally to

Schumann was steeped in romantic literature, particularly in the

He was, as we have said, an explorer and an innovator by nature;

His compositions demand from the pianist an unlimited and a

The middle section of the second movement of the great Fantasy in

Other passages offer no alternative. There is no way to suggest the

Most of Schumann’s pianoforte music is made up of short pieces.

The Carnaval, likewise a scene at a fair, representations in music of

In the Davidsbündler there is again the return at the end of familiar

The whole set presents an epitome of that imagination which gave to

As to the Sphinxes it may be said, as before, that the coherence

The Carnaval opens with a magnificent prelude. The first section of it

Pierrot and Arlequin are professional functionaries at the fair. We are

In the Carnaval, as in the Dances, Florestan breaks roughly into the

Two more professionals, masquers this time—the world-favorites,

After the waltz, a lover’s confession, hesitating but enraptured; and

There is a pause. The lovers are dispatched. Away with dreaming,

Here is a phantasmagoria unmatched elsewhere in music. It is very

The ‘Symphonic Études’ are immeasurably broader and more

There is but little trace of the composer of the Abegg variations in

The remaining movements escape language. The fifth is full of

There is a lack of titles, there is no motto, there is even no mark of

A few short pieces that Schumann published, like the Novelletten,

Four works in broad form must be reckoned among his greatest

The Fantasy is colossal. It is said that Schumann intended the first

The second movement exceeds the finale of the ‘Symphonic Études’

The concerto stands as a flawless masterpiece. The themes are

With this masterpiece we may take leave of Robert Schumann, for

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