Methods in cell biology, volume 131

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,

Methods in Cell

Biology The Neuronal Cytoskeleton, Motor Proteins, and Organelle

Trafficking in the Axon

Volume 131

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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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

Jungers Center for Neurosciences Research, Oregon Health and Science

University, Portland, OR, USA

Marvin Bentley

Jungers Center for Neurosciences Research, Oregon Health and Science

University, Portland, OR, USA

Mark M Black

Department of Anatomy and Cell Biology, Temple University School of Medicine,

Philadelphia, PA, USA

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

Kristy J Brown

Research Center for Genetic Medicine, Children’s National Health System,Washington, DC, USA; Department of Integrative Systems Biology, Institute ofBiomedical Sciences, The George Washington University, Washington, DC, USA

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

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and

the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

J.A Hammer, III

Cell Biology and Physiology Center, National Heart, Lung Blood Institute, National

Institutes of Health, MD, USA

Thomas S Hays

Department of Genetics, Cell Biology, and Development, University of Minnesota,

Minneapolis, MN, USA

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

Cell Biology, Department of Biology, Faculty of Science, Utrecht University,

Utrecht, The Netherlands

Daniel J Hu

Department of Pathology and Cell Biology, Columbia University, New York,

NY, USA

Chung-Fang Huang

Jungers Center for Neurosciences Research, Oregon Health and Science

University, Portland, OR, USA; National Laboratory Animal Center, NARLabs,

Taipei, Taiwan

Ariel Ionescu

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and

the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

Contributors xv

Kerstin M Janisch

Department of Cell Biology, University of Virginia School of Medicine,

Charlottesville, VA, USA

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,

James B Machamer

Department of Neurology, Johns Hopkins University School of Medicine,

Baltimore, MD, USA

Katalin F Medzihradszky

Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF,

San Francisco, CA, USA

Department of Anatomy and Cell Biology, University of Illinois at Chicago,

Chicago, IL, USA; Marine Biological Laboratory, Woods Hole, MA, USA

Kanneboyina Nagaraju

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

Alex V Nechiporuk

Department of Cell, Developmental and Cancer Biology, School of Medicine,

Oregon Health & Science University, Portland, OR, USA

Amanda L Neisch

Department of Genetics, Cell Biology, and Development, University of Minnesota,

Minneapolis, MN, USA

Jeffrey J Nirschl

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

Juan A Oses

Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, UCSF,

San Francisco, CA, USA

Eran Perlson

Department of Physiology and Pharmacology, Sackler Faculty of Medicine, and

the Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel

K Kevin Pfister

Department of Cell Biology, University of Virginia, Charlottesville, VA, USA

Contributors xvii

Sree Rayavarapu

Research Center for Genetic Medicine, Children’s National Health System,Washington, DC, USA; Department of Integrative Systems Biology, Institute ofBiomedical Sciences, The George Washington University, Washington, DC, USA

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

Department of Neuroscience, University of Virginia Medical School,

Charlottesville, VA, USA

Rui Yang

Jungers Center for Neurosciences Research, Oregon Health and Science

University, Portland, OR, USA

Julie Yi

Department of Pathology and Cell Biology, Columbia University, New York,

NY, USA

Wenqian Yu

Department of Neurobiology and Anatomy, Drexel University College of Medicine,

Philadelphia, PA, USA

Jie Zhou

Department of Pathology and Cell Biology, Columbia University, New York,

NY, USA

Contributors xix

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 organismsDrosophila, 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

proteins with quantum dot- or fluorochrome-labeled ligands or antibodies; andinvestigating effect of the Alzheimer’s disease peptide b-amyloid on organelletransport.

Several chapters take advantage of molecular and biochemical methods toanalyze cytoskeletal and motor protein activity The squid axoplasm system isutilized to investigate kinase pathways of phosphorylation of filament subunitsand motor proteins A proteomics method is presented to probe the effects of mousemutations on the cytoskeleton and motor proteins; and affinity chromatography isused 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 andhippocampal neurons; the second describes a plasmid-based approach to reducemyosin Va levels in cultured Purkinje cells

xxii Preface

Axonal transport: The

orderly motion of axonal

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

Axonal transport is the process by which proteins and other materials 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 Thenotion 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 bodiesdegenerate (Ramon y Cajal, 1928) However, it was not until1948that movement

synthe-of materials in axons was first revealed by Weiss and Hiscoe, who partially stricted axons and observed that axoplasm accumulated immediately proximal tothe constriction, suggesting a proximal-to-distal movement of axonal materials.Upon release of the constriction, the accumulated axoplasm moved anterogradely

transport

Since this pioneering work, axonal transport has been studied extensively withtwo experimental approaches providing most of the current understanding Oneuses radioactive precursors to pulse-label axonally transported materials and theother uses imaging techniques to directly observe transport in living axons Thesetwo approaches provide distinct but complementary information (Brown, 2009).Pulse-chase approaches provide indirect information on movement of materials inaxons 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 thecargoes that undergo axonal transport and their transport behavior as seen at shortand long time scales

The pulse-labeling approach has revealed the kinetics of protein transport in axonsover long time scales and the identity of many transported proteins Typically, radio-active amino acids are injected into the environment surrounding the neuron cellbodies under study The amino acids are taken into the neurons and incorporatedinto proteins, some of which are then transported into their axons Because theamino 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 inlength and the distribution of radioactivity along its length quantified Also, the iden-tity of specific radioactive proteins in the nerve segments has been determined usingbiochemical 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 essentialresults obtained are consistent among systems The transported pulse-labeled

2 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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 atz50e400 mm/

day (0.6e5 mm/s) (reviewed inGrafstein & 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

0.1mm/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

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 (Dahlstro¨m, 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 fromTytell et al (1981).) Panel (A) Thedistribution 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

4 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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-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:

C¼ clathrin, A ¼ actin, NFL, NFM, NFH ¼ low, middle, and heavy neurofilament subunits,

TUB¼ 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 thesedata is not straightforward First, tau, MAP1a, and MAP1b have multiple interactingpartners in addition to tubulin, some of which (e.g., actin) move in SCb, and theseinteractions can be expected to impact their movement in axons Second, live-cellimaging 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 interactionsbetween 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 sitional complexity Hundreds of diverse proteins move in SCb which include pro-teins of the actin and membrane cytoskeletons, enzymes of intermediarymetabolism, proteins involved in membrane trafficking, and proteins that interactwith synaptic vesicles Actin was one of the first proteins identified in SCb (Black

compo-& Lasek, 1979; Willard, Wiseman, Levine, compo-& Skene, 1979) It was suggested thatactin filaments form a scaffold to which other SCb proteins bind and the resultingcomplex represents an SCb cargo However, no direct data have been published tosupport this possibility

An early insight into SCb derived from the observations that SCb proteins movetogether in a vectorial manner in axons and that they are also soluble components ofaxoplasm Such a result would be difficult to explain if the proteins were freelydiffusible Thus, it was suggested that they existed as one or more assemblies thatwere conveyed by the transport machinery (Garner & Lasek, 1982; Tytell et al.,

1981) This view is supported by cell fractionation analyses which show thatmany SCb proteins behave as large multiprotein complexes (Lorenz & Willard,1978; Scott, Das, Tang, & Roy, 2011) In addition, immunoprecipitation analysesperformed 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 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 assembliesthat undergo transport in axons, the identity of these complexes remains to bediscovered

trans-This selected review has discussed some of the history that led to the structuralhypothesis of axonal transport and the initial suggestions regarding structural corre-lates of FC, SCa, and SCb Many of the suggestions were controversial sparkingnumerous studies using pulse-chase approaches that greatly enhanced knowledge

of axonal transport However, these studies did not resolve the controversy becausethey could not unambiguously reveal the identity of individual cargoes and themoment-to-moment details of their movements To move forward on these issues,

6 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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

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 movdur-ing, 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 duringtheir transport in the axon often remaining stationary for extended times and theycan also undergo changes in direction (Saxton & Hollenbeck, 2012) Yet, mitochon-dria transport is powered by the same kinesin and dynein motors that translocate FCcargoes Thus, differences in transport rate and behavior do not necessarily indicatefundamental differences in mechanism It is the differences in the regulation of thetransport machinery that allow the machinery to generate such distinctive transportbehaviors (Brown, 2003; Saxton & Hollenbeck, 2012) This same theme will come

up again in the discussion of slow axonal transport

CYTOSKELETAL POLYMERS

The first studies attempting to reveal microtubule and neurofilament transport cifically tested the hypothesis that these structures moved slowly and steadilyfrom the cell body toward the axon tip at the modal rate of SCa as revealed bypulse-chase studies (Lim, Edson, Letourneau, & Borisy, 1990; Okabe & Hirokawa,1990; Okabe, Miyasaka, & Hirokawa, 1993) The results failed to show such slowsteady movement and were interpreted as evidence that microtubules and neurofila-ments were not transported However, given that these studies failed to reveal anymovement at all including that known to occur, a more conservative interpretationwould have been that microtubules and neurofilaments do not move in a slow steadymanner As discussed below, hints already existed from pulse-chase studies on slowaxonal transport that the cargoes did not move in this manner

While pulse-chase studies showed that the bulk of neurofilament proteins movedslowly and steadily at a modal rate ofz1 mm/day, the wave is quite broad, indi-cating that some SCa cargoes move faster and some slower than this The SCawave also broadens substantially over time, further indicating that SCa cargoesmove at a distribution of rates In a particularly detailed analysis of neurofilamentprotein 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 neurofilamentproteins moved with brief but rapid translocation steps interrupted by pauses This

is similar to the situation for mitochondria, but with pauses accounting for amuch greater percentage of the transport behavior to account for the slow averagerate of neurofilament protein transport Although speculative at the time, this viewpresaged 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 rofilament transport in living axons, followed shortly thereafter byRoy et al (2000)

neu-8 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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 unexpecinterrup-tedly, 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 Overthe 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

Several studies have demonstrated that microtubules can redistribute within growingneurons 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 accountedfor 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 methodsclearly revealed tubulin moving in living axons, and like neurofilaments, the tubulin-containing cargo moved rapidly but intermittently with an average rate in the rangereported for tubulin transport as seen in the pulse-chase studies (Hasaka, Myers, &Baas, 2004; He, Francis, Myers, Black, & Baas, 2005; Wang & Brown, 2002) Themovement 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(average¼ 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 suggestedthat 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 croscopy to reveal individual axonal microtubules in living axons, and none of thesepolymers was observed to move In another approach, microtubule plus ends weretagged with fluorescent tip-binding proteins and then imaged to see whether thepolymers moved It is expected that the plus ends will advance as the microtubuleselongate If they also undergo transport, then the rate of advance will exceed that due

mi-to microtubule elongation alone However, in no case was this observed (Kim &Chang, 2006; Ma, Shakiryanova, Vardya, & Popov, 2004) As these studies imagedlarge 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 restrictanalyses 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 bythe 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 movingtubulin-containing structures are in fact short microtubules Given that tubulin

10 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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 byCampenot 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 (Dz 3 mm2/s) and

that tau association with microtubules exhibits a high exchange rate (t1/2z 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 thegaps The movement occurred in both anterograde and retrograde directions andexhibited stop-and-go characteristics with brief bouts of fast transport (0.2e

2mm/s) interrupted by pauses The transport of these tau-containing structuresstrikingly resembles that of tubulin-containing structures It is noteworthy thatthe manipulation that led to the detection of tau transport specifically involvedincreasing the number of microtubule-binding domains on tau Thus, the mostparsimonious explanation of the data on tubulin and tau transport is that tubulin

is transported as short microtubules and tau transport reflects its association withtransported microtubules

It has been argued that microtubule transport is important for establishing themicrotubule polarity pattern of the axon, for the expansion of the axonal microtubulearray during growth and development and its maintenance in the adult (Baas, 2002;Baas & Ahmad, 1993; Black, 1994) Impairment of microtubule transport in axonsmay also be a factor in neurodegenerative diseases by compromising the axonalmicrotubule 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, someuncertainty remains It is imperative to directly test whether these tubulin-containingstructures are indeed microtubules and hopefully move past this lingering uncer-tainty Methods are available for doing this using reagents that both fluoresce andcan be seen using the electron microscope While such experiments pose technicalchallenges, the effort will be worth the outcome because the issue will be resolvedonce and for all, and the outcome will provide essential direction for the field tomove forward

AXOPLASM

In some respects, little progress has been made in deciphering SCb, whereas in otherrespects great strides have been made With regard to the former, to explain the

assemble into multiprotein complexes that are the cargoes of SCb (Garner & Lasek,1982; Lorenz & Willard, 1978) While recent studies have provided further supportfor the hypothesis that SCb proteins assemble into multiprotein complexes (Scott

et al., 2011; Tang, Das, Scott, & Roy, 2012), the identity of the complexes and theirpossible relationship to cytological structures of the axon remain unknown On theother hand, substantial progress has been made in dissecting the transport mecha-nisms for these proteins As described below, the theme of rapid but infrequentmovements also figures important for SCb

Initial studies expressed GFP-tagged SCb proteins (a-synuclein, synapsin-1,glceraldehyde-3-phosphate dehydrogenase) in cultured hippocampal neurons, andused imaging parameters to detect rapid but intermittent movements (Roy, Winton,

12 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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

2mm/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-taggeda-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

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 ofz0.01 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 ofthe bulk of SCb proteins as well as the more persistent particle movements, focusingspecifically 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 arange 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 interactionstrengths, such that most movements were of short duration (1 s) and distance(1 mm), although a minor fraction persisted for many seconds and moved manymicrons Given the biochemical evidence suggesting that synapsin-1 along withother SCb proteins exist as multiprotein assemblies, this suggests a model in whichcomplexes of SCb proteins containing synapsin-1 transiently engage with motors,either directly or indirectly, resulting in an overall slow anterograde advance withinthe axon.

Given the dual imaging analyses showing that SCb cargoes are cotransportedwith synaptophysin (Roy et al., 2007; Scott et al., 2011), the vesicular cargoes of

FC that contain synaptophysin are logical candidates for the mobile units Directsupport 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 vesicularstructures Refinements of the simulation parameters suggest a model in which syn-apsin-1 assembles into multiprotein complexes that have an affinity to vesicularcargoes of fast transport The synapsin-1 complexes and vesicles interact stochasti-cally, with most synapsin-1 complexes interacting transiently and thus advancingslowly 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 plexes (Black et al., 1991; Garner & Lasek, 1982; Lorenz & Willard, 1978; Roy

com-et al., 2007) A number of SCb proteins are able to interact directly or indirectlywith membranes (for example, a-synuclein, spectrin, actin, clathrin) and so maymove 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 bymolecular motors in a manner that results in an overall slow transport within theaxon 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 tery Many of its proteins still remain to be identified and the current understanding

mys-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 vided a mechanistic understanding of the transport of select SCb proteins and thenext several years promise to reveal many new insights into this still enigmaticcomponent of axonal transport

pro-14 CHAPTER 1 Axonal transport: The orderly motion of axonal structures

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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Yan, Y., & Brown, A (2005) Neurofilament polymer transport in axons Journal of

Neuroscience, 25, 7014e7021

Yu, W., Schwei, M J., & Baas, P W (1996) Microtubule transport and assembly during axon

growth Journal of Cell Biology, 133, 151e157

Live-cell imaging of

neurofilament transport in

Atsuko Uchida, Paula C Monsma, J Daniel Fenn, Anthony Brown 1

Department of Neuroscience, The Ohio State University, Columbus, OH, USA

1 Corresponding author: E-mail: brown.2302@osu.edu

1.2.1 Media for superior cervical ganglion neuron cultures 26

1.2.2 Media for cortical neuron cultures 27

1.2.3 Media for dorsal root ganglion neuron cultures 28

1.3 Coverslips and Culture Dishes 29

1.3.1 Glass-bottomed dishes 30

1.3.2 Sterilizing and coating coverslips and glass-bottomed dishes 32

1.4 Superior Cervical Ganglion Neuron Cultures 34

1.5 Cortical Neuron Cultures 36

1.5.1 Preparation of coverslips for glial sandwich cultures 37

1.5.2 Preparation of a suspension of cortical neurons and glia 37

1.5.3 Preparation of cortical neuron cultures 40

1.5.4 Preparation of glial sandwich cultures 40

1.6 Dorsal Root Ganglion Neuron Cultures 42

1.6.1 Long-term myelinating DRG cocultures 42

1.6.2 Short-term nonmyelinating DRG cultures 44

2 Neurofilament Fusion Proteins 44

2.1 Choice of Neurofilament Subunit 44

2.2 Choice of Fluorescent Protein 45

2.3 Design of Fusion and Expression Construct 46

2.4 Testing the Constructs for Assembly Competence 48

3 Transfecting Neurons 49

3.1 General Considerations 49

3.2 Plasmid Purification 49

CHAPTER

Methods in Cell Biology, Volume 131, ISSN 0091-679X, http://dx.doi.org/10.1016/bs.mcb.2015.07.001

3.3 Electroporation 493.4 Lipofection 523.5 Magnetofection 523.6 Nuclear Injection 53

4 Strategies for Observing Movement 57

4.1 General Considerations 574.2 Naturally Occurring Gaps 584.3 Fluorescence Photobleaching 614.4 Fluorescence Photoactivation 61

5 Live-Cell Imaging 65

5.1 General Considerations 655.2 Imaging Media 665.3 Controlling Temperature, CO2, and Humidity 665.3.1 Stage-top incubators 665.3.2 Imaging chambers 675.3.3 Air stream incubators 685.3.4 Objective heaters 685.4 Choice of Microscope 685.5 Choice of Objective 695.6 Choice of Camera 705.7 Selection of Cells and Axonal Regions for Imaging 715.8 Time-Lapse or Streaming Image Acquisition 725.9 Focus Drift 735.10 Minimizing Photobleaching and Photodamage 745.11 Methods for Spatially Selective Illumination 745.11.1 Iris diaphragm method 755.11.2 DMD method 755.11.3 Laser scanning method 765.12 Fluorescence Photobleaching Experiments 765.13 Fluorescence Photoactivation Experiments 77

6 Software and Analysis 78

6.1 Software 786.2 Fixed-Field Tracking 796.3 Multifield Tracking 816.4 Kymograph Analysis 826.5 Pulse-Escape Fluorescence Photoactivation Analysis 83

7 Summary 86 Acknowledgments 87 References 87

Neurofilaments, which are the intermediate filaments of nerve cells, are space-filling

cytoskeletal polymers that contribute to the growth of axonal caliber In addition to their

structural role, neurofilaments are cargos of axonal transport that move along microtubule

tracks in a rapid, intermittent, and bidirectional manner Though they measure just 10 nm

in diameter, which is well below the diffraction limit of optical microscopes, these

polymers can reach 100mm or more in length and are often packed densely, just tens of

nanometers apart These properties of neurofilaments present unique challenges for studies

on their movement In this article, we describe several live-cell fluorescence imaging

strategies that we have developed to image neurofilament transport in axons of cultured

neurons on short and long timescales Together, these methods form a powerful set of

complementary tools with which to study the axonal transport of these unique intracellular

cargos

List of Abbreviations

INTRODUCTION

Neurofilaments, which are the intermediate filaments of nerve cells, are long flexible

rope-like cytoskeletal polymers composed of internexin, peripherin, and

neurofila-ment proteins L, M, and H (low, medium, and high molecular mass) (Laser-Azogui,

Kornreich, Malka-Gibor, & Beck, 2015; Perrot & Eyer, 2013) Like other

interme-diate filament proteins, these proteins all share a conserved alpha-helical central

domain, called the rod domain, that assembles to form the filament backbone,

flanked by variable amino- and carboxy-terminal domains which regulate subunit

assembly and filament interactions However, neurofilament proteins can

coassem-ble in various combinations and stoichiometries so the precise composition of

neuro-filaments depends on which of the neurofilament proteins are expressed and on their

Introduction 23

relative level of expression, both of which vary depending on the neuronal cell typeand stage of development.

The principal known function of neurofilaments is to expand axon caliber, which

is a major determinant of axonal conduction velocity (Hoffman, 1995) The filling properties of these polymers are maximized by the carboxy-terminal domains

space-of the subunit proteins, particularly space-of neurspace-ofilament proteins M and H, which ect outward from the filament backbone The extended and unstructured nature ofthese highly charged domains forms a dense polyelectrolyte brush border that ap-pears to define a zone of exclusion around the backbone of the polymer muchlike the bristles on a bottle brush, increasing the spacing between neurofilamentsand neighboring structures (Mukhopadhyay, Kumar, & Hoh, 2004)

proj-In addition to their structural role, neurofilaments are cargos of axonal transportthat move along microtubule tracks, propelled by microtubule motor proteins (Brown,

2000, 2009; Wang, Ho, Sun, Liem, & Brown, 2000) The observed rate of movementdepends on the time frame of observation Radioisotopic pulse-labeling experiments

in laboratory animals on a timescale of days or weeks have revealed that ments are conveyed in the slow component of axonal transport at average rates ofapproximately 0.3e3 mm/day In contrast, live-cell fluorescence imaging in culturedneurons on a timescale of seconds or minutes has revealed that neurofilaments move

neurofila-at average rneurofila-ates ofw1e3 mm/s, but these rapid bouts of movement are intermittentand highly asynchronous Thus, the axonal transport of neurofilaments is a “stop-and-go” motility characterized by rapid infrequent movements interrupted by pro-longed pauses The velocity is slow on long timescales because the polymers spendmost of their time pausing It is now generally assumed that other cargos of slowaxonal transport are transported in a similar stop-and-go manner, but this has yet

to be proven

Neurofilaments are also of clinical interest because mutations in neurofilamentprotein L (NFL) can cause peripheral neuropathy and because neurofilaments accu-mulate abnormally in many different neurodegenerative diseases and toxic neurop-athies (Lariviere & Julien, 2004) In extreme cases, the neurofilament accumulationslead to large balloonlike swellings of the affected axons These accumulations arethought to be caused by an impairment of the axonal transport of these cytoskeletalpolymers, but how this occurs is poorly understood

In spite of considerable progress over the past 15 years, there are still many swered questions regarding the mechanism of neurofilament transport For example,how do motors interact with neurofilaments, how do these motors coordinate bidi-rectional motility, how is the organization of neurofilaments in axons establishedand maintained, what determines when neurofilaments move and pause, what regu-lates the accumulation of neurofilaments in developing axons, and what causes neu-rofilaments to accumulate abnormally in axons in neurodegenerative disease?Cultured neurons offer researchers a special opportunity to address these questionsbecause of their amenability to observation by high-resolution live-cell fluorescenceimaging However, a major challenge is that these polymers measure just 10 nm indiameter and are often packed close together, just tens of nanometers apart Since the

unan-diameter and spacing of these polymers are well below the optical diffraction limit

of light microscopes, the movement of single neurofilament polymers can be

diffi-cult to detect A further challenge is that the movement is very infrequent, with

sin-gle neurofilaments capable of pausing for hours between successive bouts of

movement In this article, we describe fluorescence imaging strategies that we

have developed to overcome these and other challenges Each of these approaches

has its strengths and weaknesses, but taken together they form a set of powerful

and complementary tools with which to investigate the kinetics of neurofilament

transport in a variety of neuronal cell types

We prefer to use primary neuronal cultures, which are cultures of differentiated

nerve cells isolated from neuronal tissue Since these neurons are postmitotic

differ-entiated cells, each batch of cultures yields a fixed number of neurons Typically, we

establish cultures on a weekly or biweekly basis depending on our needs For our

live-cell imaging studies of neurofilament transport we have used autonomic motor

neurons from superior cervical ganglion (SCG), sensory neurons from dorsal root

ganglion (DRG), or neurons from cerebral cortex (“cortical neurons”) We establish

these cultures from either rats or mice We purchase timed pregnant rats or mice

from Harlan (Indianapolis, IN) or Taconic Biosciences (Cambridge City, IN) If

we have a choice we generally prefer to use rats, but in general the procedures

are identical for mice We focus here on our own current methods Similar or

alter-native methods for culturing these neuronal cell types, which include detailed

descriptions of the tissue dissection and culturing procedures, can be found

else-where (Goslin, Hannelore, & Banker, 1998; Hawrot & Patterson, 1979; He &

Baas, 2003; Higgins, Lein, Osterhout, & Johnson, 1991; Johnson & Argiro, 1983;

Johnson, Iacovitti, Higgins, Bunge, & Burton, 1981; Kaech & Banker, 2006;

Kleitman, Wood, & Bunge, 1998; Mahanthappa & Patterson, 1998)

To establish and maintain primary neuronal cultures, we use fine dissecting

in-struments, a binocular dissecting microscope capable of 8e25x magnification, a

horizontal laminar flow hood, and a tissue culture incubator with temperature and

CO2 control as well as active or passive humidification The fine dissection and

all subsequent steps are performed under sterile conditions in the hood to avoid

bac-terial or fungal contamination (Freshney, 2010, 796 pp.) Hoods with a vertical flow

design are not suitable because they cannot accommodate a dissection microscope

With the exception of myelinating cocultures (Section 1.6), we generally prefer

to plate the cells at low densities At higher densities, the axons tend to fasciculate,

which makes axon tracing more challenging In addition, axons in dense cultures

often do not lie directly against the glass coverslip, making it hard to keep moving

neurofilaments in focus Higher cell densities also result in more nonneuronal cells

1 Culturing neurons 25

and more cell debris, leading to higher background fluorescence However, sinceneurons fare much better in high-density cultures, culturing them at low densityrequires that extra care and attention be paid to the culture procedures and condi-tions We maintain the cultures in an incubator at 37
C and 95% relative humidity

in an atmosphere of either ambient or 5% CO2, depending on the culture medium

1.2.1 Media for superior cervical ganglion neuron cultures

For short-term cultures (up to 1 week or so), we use a medium based on the

red-free) supplemented with 10% (v/v) adult rat serum, 0.6% (w/v) glucose,

50 ng/mL nerve growth factor (NGF; 2.5S subunit purified from mouse salivaryglands) (L-15 culture medium; Table 1) L-15 medium is designed to buffer its

pH at atmospheric CO2so it is more convenient for observation and microinjection

of cells on the microscope stage than media with bicarbonate buffering systems Anadditional benefit is that nonneuronal cells proliferate far less in this medium than in

Table 1 Composition of SCG Neuron Culture Media The L-15 culture medium is

maintained at atmospheric CO2and the DMEM/F12 culture medium is maintained at5% CO2 DMEM/F12 is a 1:1 (V/V) mixture of Dulbecco’s Modified Eagle and Ham’sF-12 nutrient media 100mg/mL bovine transferrin can be substituted with 20 mg/mLrat transferrin

L-15 Culture medium

Leibovitz’s L-15 medium, phenol red-free Gibco Life Technologies

0.5% (w/v) Methocel Ô (F4M premium grade) Dow Chemical Company

DMEM/F12 Culture medium

DMEM/F12 medium, phenol red-free Gibco Life Technologies

0.5% (w/v) bovine serum albumin, fraction V EMD Millipore

0.5% (w/v) Methocel Ô (F4M premium grade) Dow Chemical Company

bicarbonate-buffered media, thereby eliminating the need for the addition of

antimi-totic agents NGF is required to support the survival of the neurons For longer term

cultures, we have used a serum-free DMEM/F12-based medium based on the N2

formulation ofBottenstein and Sato (1979) as modified by Higgins et al (1991)

(DMEM culture medium;Table 1) For low-density cultures, this medium can be

supplemented with 10% adult rat serum A defined serum-free L-15-based culture

medium can also be used (Hawrot & Patterson, 1979; Mahanthappa & Patterson,

1998) With good sterile technique it is not necessary to use antibiotics, which

can have deleterious side effects on neurons

Adult rat serum can be prepared by the method ofHawrot and Patterson (1979)

or purchased from a commercial source We sterilize the serum by syringe filtration

with a 0.2mm filter and store it frozen in aliquots to minimize repeated freezing and

thawing Often the serum forms a fine precipitate after a few days at 37
C This

pre-cipitate can be mistakenly identified as microbial contamination, but it is innocuous

and does not harm the cells With serum from some commercial sources, the

precip-itate can become so dense that it obscures the cells In this case, switch to a different

commercial source or prepare the serum yourself Fetal bovine serum (FBS) can also

be used, but it is our impression that both rat and mouse SCG neurons are healthier

and attach better to the substrate in the presence of adult rat serum

The function of the MethocelÔ is to increase the viscosity of the culture

me-dium It is not critical, but the cells appear to attach better to the substrate when it

is present Since MethocelÔ solutions are too viscous to sterilize by filtration, the

in 50 mL disposable polypropylene centrifuge tubes and then autoclave with the

caps loosely attached After cooling, the caps can be tightened, and the tubes can

be stored indefinitely at room temperature To dissolve the MethocelÔ, we add

40 mL sterile L-15 and shake the tube overnight at 37
C The resulting solution

contains insoluble particulate material, which can be removed by filtration using a

5mm syringe filter (Millex-SV, EMD Millipore)

1.2.2 Media for cortical neuron cultures

The cortical neurons are cultured with a glial feeder layer To expand and maintain the

glia, we use a medium consisting of Minimum Essential Medium (MEM)

supple-mented with 10% (v/v) horse serum, 0.7% (w/v)D-glucose, and 0.5mg/mL gentamicin

medium (BrainBits LLC, Springfield, IL) with or without additional salt depending

on the desired osmolarity (NbActiv4Ô culture medium; Table 2) NbActiv4Ô is

identical to Neurobasal/B27Ô culture medium except for three additional

supple-ments: creatine, estrogen, and cholesterol NbActiv4Ô medium has been reported

to yield hippocampal neuron cultures with more synapses, increased electrical

activ-ity, and less metabolic stress compared to Neurobasal/B27Ô medium (Brewer et al.,

2009) In our experience, we obtain improved cell health and viability using this

medium To enhance neuronal recovery improve cell attachment and minimize the

risk of bacterial contamination arising from the initial dissection, we supplement

1 Culturing neurons 27

the NbActiv4Ô medium with 5% (v/v) FBS and 0.5 mg/mL gentamicin for plating

the cultures in this medium With good sterile technique the gentamicin can beomitted Around 1 week after plating, we increase the osmolarity of the medium

concentra-tion of 37.5 mM, which we have found to improve long-term cell viability (Section5.2) While it is possible to culture cortical neurons in this medium for at least

1 month, all of our own work has been performed within 2 weeks of plating

1.2.3 Media for dorsal root ganglion neuron cultures

For long-term myelinating cocultures of DRG neurons and Schwann cells, we use

with SCG neurons, a neurotrophic factor is required to support neuron survival.NGF supports the survival of cutaneous sensory neurons, which have relativelysmall axon diameters, whereas NT-3 supports the survival of the proprioceptivesensory neurons that innervate the skeletal muscles The NT-3-dependent neuronscontain more neurofilaments, have larger axons, and myelinate more readily andmore continuously in culture Five days after plating the cells, the medium isreplaced with fresh medium containing a one-time supplement of MatrigelÔ at a1:100 dilution (BD Biosciences) Three days later, half the medium is removed

Table 2 Composition of Cortical Neuron and Glia Culture Media These media are

maintained at 5% CO2

Glia MEM Culture medium

Minimum Essential Medium (MEM) Gibco Life Technologies

10% (v/v) horse serum Gibco Life Technologies

0.7% (w/v) D -glucose (39 mM) Sigma

NbActiv4 Ô Plating medium

5% (v/v) fetal bovine serum (FBS) Hyclone, GE Healthcare Life Sciences

NbActiv4 Ô Culture medium

37.5 mM NaCl (included w1 week after

plating)

Sigma