Force generation: ATP-powered proteasomes pull the rope.

Abstract

Recently, single-molecule force spectroscopy techniques have provided unprecedented opportunities to apply and to quantify forces that guide protein (un-)folding. A new study provides fascinating insights into the sophisticated mechanism by which an ATP-fueled proteolytic machine generates mechanical forces to unfold and translocate multidomain substrates.

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of random connections as one
possible explanation for their finding.
But to suppose that the sophisticated
neural functions that are needed
even before birth arise magically by
experience-dependent re-organization
of an initial network of random
connections flies in the face of
evidence and of logic. Tabula rasa
has not been a serious hypothesis
since Ramon y Cajal first observed
that nervous systems consist of
neurons that form specific connections
from the earliest outgrowth of their
axons and dendrites. The studies of
Bock et al. [3] and Ko et al. [4] support
his notion of specificity. The ghost of
tabula rasa should be laid to rest for
once and for all.
It is certainly not a general property of
all inhibitory celltypesthattheyconnect
to all excitatory cell targets in their
vicinity or vice versa [5]. Thus, the
densely connected inhibitory neurons
observed in two of the studies reviewed
here should not be taken as evidence of
‘promiscuous innervation’ as Fino and
Yuste [1] describe it, but of a deliberate
wiring strategy. What we do not yet
understand is why the wiring is like it is.
Fino and Yuste [1] speculate that their
inhibitory circuit might perform the
housekeeping function of keeping
excitation within some operating
range. Taken together, however, the
three studies reveal circuits with
much richer possibilities for
computation. For example, the patterns
of connections reported are consistent
with the ‘ring-of-neurons’ model
(Figure 2), which can generate a number
of critical computational ‘primitives’ [6],
including the ‘soft’ Winner-Take-All
operationimplicatedinsuchkeycortical
operations as selective amplification,
signal restoration, and decision-making
(see, for example, [6–8]).
Then there is the matter of the
‘unknown’ connectivity diagram. While
it is true that Ramon y Cajal [2] failed to
describe a connectivity diagram for
neocortex, it is also true that since the
1970s at least there have been any
number of diagrams that,despite being
drawn from different cortical areas and
different species, show such family
resemblances that we have suggested
these might be ‘canonical’ circuits for
neocortex [5]. Our hope is that these
new tools will provide the means of
exploring many more cortical areas in
detail, rather than the one or two that
dominate current studies.
The twenty-first century has given us
something that the Golgi technique
never could — quantitative connection
maps. To the question, who connects
to whom, we can now add, ‘and how
much?’ The current enthusiasm for
connectivity diagrams is clearly
infectious, but these diagrams are only
a necessary, not sufficient, condition
for understanding the principles
organising cortical circuits and their
function. As Horace Barlow elegantly
put it [9]: ‘‘We badly need all possible
information on what one might call
‘principles and technology of neural
engineering’ and the only way to
acquire it is to relate anatomical
structures and cellular function to
overall performance.’’ The
technologies on display here are
convincing evidence that we have
never been better equipped todiscover
these principles.
References
1. Fino, E., and Yuste, R. (2011). Dense inhibitory
connectivity in neocortex. Neuron 69,
1188–1203.
2. DeFelipe, J., and Jones, E.G. (1988). Cajal on the
Cerebral Cortex (New York: Oxford University
Press), pp. 654.
3. Bock, D.D., Lee, W.-C.A., Kerlin, A.M.,
Andermann, M.L., Hood, G., Wetzel, A.W.,
Yurgenson, S., Soucy, E.R., Kim, H.S., and
Reid, R.C. (2011). Network anatomy and in vivo
physiology of visual cortical neurons. Nature
471, 177–182.
4. Ko, H., Hofer, S.B., Pichler, B., Buchanan, K.A.,
Sjo ¨stro ¨m, P.J., and Mrsic-Flogel, T.D. (2011).
Functionalspecificityoflocalsynapticconnections
in neocortical networks. Nature 473, 87–91.
5. Douglas, R.J., and Martin, K.A.C. (2004).
Neuronal circuits of the neocortex. Annu. Rev.
Neurosci. 27, 419–451.
6. Douglas, R.J., and Martin, K.A.C. (2007).
Recurrent neuronal circuits in the neocortex.
Curr. Biol. 17, R496–R500.
7. Douglas, R.J., Koch, C., Mahowald, M.,
Martin, K.A.C., and Suarez, H.H. (1995).
Recurrent excitation in neocortical circuits.
Science 269, 981–985.
8. Rutishauser, U., Douglas, R.J., and Slotine, J.J.
(2011). Collective stability of networks of
winner-take-allcircuits.NeuralComp.23,735–773.
9. Barlow, H.B. (1977). Performance, perception,
dark-light, and gain boxes. In Neuronal
Mechanisms in Visual Perception, E. Po ¨ppel,
R. Held, and J.E. Dowling, eds. (Cambridge:
Neurosciences Research Program Bulletin 15,
MIT Press), (1977) pp. 394–397.
Institute of Neuroinformatics, UZH/ETH,
Winterthurerstrasse 190, 8057 Zurich,
Switzerland.
E-mail: kevan@ini.phys.ethz.ch
DOI: 10.1016/j.cub.2011.04.026
Force Generation: ATP-Powered
Proteasomes Pull the Rope
Recently, single-molecule force spectroscopy techniques have provided
unprecedented opportunities to apply and to quantify forces that guide protein
(un-)folding. A new study provides fascinating insights into the sophisticated
mechanism bywhich an ATP-fueled proteolytic machine generates mechanical
forces to unfold and translocate multidomain substrates.
Yves F. Dufre ˆne1and Daniel J. Mu ¨ller2
The force-induced conformational
changes of cellular proteins play major
roles in mediating physiological
functions. Prominent examples are
mechanosensors, which convert
mechanical forces into biochemical
signals, cell-adhesion proteins such
as integrins, which strengthen and
regulate cell adhesion through
force-induced functional states, and
proteases such as ATP-dependent
proteolytic machines, which recognize,
unfold and translocate specific protein
substrates. In a recent issue of
Cell, Aubin-Tam et al. [1] use
single-molecule force spectroscopy
(SMFS) to study how the ClpXP
protease generates force to unfold
and translocate multidomain
substrates.
The past several years have
witnessed remarkable progress in
applying SMFS to measure the
molecular force response of proteins
[2–6]. SMFS describes a family of
related techniques that apply and
measure forces over different
length (z0.1 nm to 100 mm) and/or
time (z0.1 ms to 100 s) scales.
SMFS-based assays measure
forces exerted on — and generated
by — single molecules over a wide
range of forces (from z0.1 pN to
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100 nN) that are relevant to biology,
ranging from weak intermolecular and
intramolecular interactions to strong
covalent bonds and complex cellular
interactions. The SMFS family includes
atomic force microscopy (AFM),
biomembrane force probe (BFP),
microneedles, and magnetic and
optical tweezers. Whereas AFM, BFP
and microneedles measure molecular
forces using mechanical force
transducers, optical and magnetic
tweezers use external electromagnetic
fields.
SMFS experiments have
impressively demonstrated the crucial
role of protein mechanics in
biochemistry and cell biology [2,6]. The
general idea behind these studies is to
characterize how single proteins react
upon exposure to an external force.
The first such example was the
mechanical unfolding of the multiple
immunoglobulin domains of the giant
protein titin: upon stressing the
multidomain construct with sufficiently
high force, one immunoglobulin
domain unfolded after the other until all
domains were unfolded. However,
SMFS studies have also revealed that
mechanical stimuli can alter protein
conformation and activity by
various mechanisms, including: the
force-induced exposure of cryptic
peptide sequences or catalytic
sites, such as in fibronectin and
integrins [2–5,7]; the opening of
mechanosensitive ion channels [8];
the strengthening of receptor–ligand
interactions by tensile mechanical
force, such as catch bonds mediated
by bacterial adhesive proteins [9]; and
the binding of cytoskeletal proteins
(like vinculin) to mechanically stretched
cytoplasmic proteins (like talin rod
molecules) [10]. In addition, SMFS has
provided key insights into how motor
proteins walk along the cytoskeleton
and transport cargo, and into the
translocation and packaging
mechanism of nucleic acid binding
motor proteins [11,12]. In the new
study, Aubin-Tam et al. [1] nicely
unravel how an ATP-fueled proteolytic
machine develops mechanical force to
drive protein unfolding and
translocation (Figure 1A).
Living cells use ATP-powered
proteases for protein quality control
and regulation. A prominent example
is the ClpXP AAA+ protease complex,
in which ClpX recognizes, unfolds, and
then translocates proteins into ClpP for
degradation.Bothenzymescombineto
form a hexameric ring of ATPases and
a barrel-like peptidase, in which the
proteolytic active sites are concealed
within an interior chamber [1].
Degradation is initiated when loops
within the axial pore of the ClpX ring
engage an unstructured region of
a protein substrate. ATP-fueled
conformational changes in the ClpX
ringare then thought toproduce pulses
of pulling that unfold the protein
substrateandtranslocate itthroughthe
pore and into the degradation chamber
of ClpP [13]. Until now, however, the
mechanisms underlying protein
unfolding and degradation were poorly
understood at the molecular level.
Aubin-Tam et al. [1] used SMFS to
measure the mechanics of enzymatic
unfolding and translocation of single
ClpXP machines of a multidomain
substrate. To probe single-molecule
mechanical activity, they tethered
a complex of ClpXP and a multidomain
substrate containing eight distinct
immunoglobulin-like domains of the
naturally occurring filamin A protein
between two polystyrene beads, held
in a dual laser-trap configuration
(Figure 1A). The traps were adjusted to
maintain a constant tension and to
allow the ClpXP bead to move towards
the other bead to compensate for
changes in substrate length. As shown
in Figure 1B, ClpXP-mediated
unfolding of a domain of the substrate
increases the bead–bead distance,
whereas translocation of the unfolded
polypeptide decreases this distance.
The dwell time is the time required to
unfold the next domain. Using this
single-molecule assay, the authors
demonstrated that ClpXP sequentially
unfolds and translocates each
Forced unfolding
Distance
ClpX
Distance
Multidomain substrate
DNA
linker
A
Translocation
Distance
B
Time
Current Biology
Distance
Unfolding
Dwell time
Translocation
Time
ClpP
ClpXP
ClpXP
F
F
F
F
F
F
Figure 1. Single-molecule force spectroscopy (SMFS) unravels the role of protein mechanics
in biology.
In vitro SMFS reveals the mechanics of ClpXP-mediated protein unfolding. (A) Top: experi-
mental design showing the optical double-trap device in which ClpXP was attached to one
polystyrene bead (gray), while a multidomain substrate was tethered to a second bead by
double-stranded DNA. As soon as the substrate is threaded through the ClpX pore, ATP-pow-
ered conformational changes of loops unfold each domain. Middle: ClpXP exerts a force to
induce the unfolding of a domain of the substrate, increasing the distance between both
beads. Middle and bottom: translocation of the unfolded polypeptide through ClpXP proceeds
and it is degraded into small fragments in the lumen of ClpP. The distance between the beads
is consequently reduced with respect to the starting distance. F, force. (B) Schematic of
a representative extension-versus-time trace of unfolding and translocation events. The dwell
time refers to the time required to unfold the next domain of the substrate.
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successive domain of this substrate in
a highly localized and unidirectional
fashion, and that unfolding of individual
substrate domains is very fast (<1 ms)
and highly cooperative.
A remarkable finding of this study is
the relatively small translocation step
size (5–8 amino acids) of ClpXP,
indicating a low gear that can work
against substantial force loads [1].
As observed for other motor proteins,
the translocation velocity of ClpXP
depends on the force load that
stretched the substrate. Each step
of ClpXP translocation against
20 pN of applied load corresponds to
5 kT of mechanical work, which
requires energetically costly ATP
hydrolysis events. The data support
a power-stroke model of denaturation
in which successful enzyme-mediated
unfolding of stably folded domains
requires coincidence between
mechanical pulling by the enzyme
and a transient stochastic reduction in
protein stability. With increasing
stability of the substrate protein, the
ATP consumption of the process
increases. The process of repeatedly
pulling a peptide end is a simple
mechanical mechanism that allows
relatively low-cost denaturation of
metastable proteins but high-cost
denaturation of very stable proteins.
Because ClpXP and other AAA+
proteases denature a highly diverse
assortment of cellular proteins
exposing very different structures and
stabilities, it is unlikely that evolution
has optimized the protease activities
for any single substrate. However,
some AAA+ proteases are specialized
to degrade poorly structured proteins
and, thus, show limited unfoldase
activity. Because the energy
consumption of proteases increases
with translocation step size, it will be
interesting to characterize whether
these proteases consume less energy
for degradation.
In conclusion, this elegant study
demonstrates how ATP-powered
proteases are able to generate high
forces to carry out mechanical work in
order to achieve function, i.e. the
unfolding and degradation of specific
proteins. What are the challenges
ahead? Like most single-molecule
force experiments, this study was
conducted in vitro, i.e. on isolated
molecules where the biological system
can be tightly controlled. However, the
interactions guiding the assembly and
functional state of the biomolecular
machinery are dynamically controlled
by the living cell, emphasizing the need
to move single-molecule experiments
into living cells [6,14]. Although in most
examples SMFS isused to characterize
one protein interacting with another, it
is clear that in a cell there are many
more interacting partners (Figure 2).
It is also clear that, when extracting
two cellular interaction partners, their
possible interaction pathways have
been in most cases significantly
narrowed. Today, we must use such
a reductionist approach to study the
biomolecular machinery of the cell
because the current single-molecule
technology cannot yet be brought into
living cells. In the future, overcoming
the problems and challenges to
transfer SMFS technologies into the
cell will allow us to learn how the
cellular machinery is controlled in vivo
and to understand how the living cell
uses forces to guide its biomolecular
machinery.
References
1. Aubin-Tam, M.E., Olivares, A.O., Sauer, R.T.,
Baker, T.A., and Lang, M.J. (2011). Single-
molecule protein unfolding and translocation
by an ATP-fueled proteolytic machine. Cell 145,
257–267.
2. Bustamante, C., Macosko, J.C., and
Wuite, G.J.L. (2000). Grabbing the cat by the
tail: manipulating molecules one by one. Nat.
Rev. Mol. Cell Biol. 1, 130–136.
3. Vogel, V., and Sheetz, M. (2006). Local force
and geometry sensing regulate cell functions.
Nat. Rev. Mol. Cell Biol. 7, 265–275.
4. Sotomayor, M., and Schulten, K. (2007).
Single-molecule experiments in vitro and
in silico. Science 316, 1144–1148.
5. Brown, A.E.X., and Discher, D.E. (2009).
Conformational changes and signaling in cell
and matrix physics. Curr. Biol. 19, R781–R789.
6. Mu ¨ller, D.J., Helenius, J., Alsteens, D., and
Dufre ˆne, Y.F. (2009). Force probing surfaces of
living cells to molecular resolution. Nat. Chem.
Biol. 5, 383–390.
7. Friedland, J.C., Lee, M.H., and Boettiger, D.
(2009). Mechanically activated integrin switch
controls a5b1 function. Science 323, 642–644.
8. Li, Y., Wray, R., Eaton, C., and Blount, P. (2009).
Anopen-porestructureofthemechanosensitive
channel MscL derived by determining
transmembrane domain interactions upon
gating. FASEB J. 23, 2197–2204.
9. Yakovenko, O., Sharma, S., Forero, M.,
Tchesnokova, V., Aprikian, P., Kidd, B.,
Mach, A., Vogel, V., Sokurenko, E., and
Thomas, W.E. (2008). FimH forms catch bonds
Signal
pathway
Dynamic
reassembly
and functional
regulation of the
cytoskeleton
F
Functional
regulation
F
F
F
F
F
F
F
F
F
F
Cell–cell
adhesion
F
Protein
unfolding
and
degradation
Transport
DNA
DNA
motors
Membrane
shaping
F
F
Figure 2. Bringing the SMFS toolbox into living cells.
In the future, in vivo SMFS will offer a means to force-probe the mechanics and interactions
that drive the molecular machinery of the cell, including: the dynamic assembly of supramo-
lecular complexes; transport phenomena; protein folding, unfolding and degradation;
membrane protein insertion and folding; membrane shaping and reorganization; DNA-binding
motor proteins; cell adhesion and signaling; signaling pathways; and interactions of the
cytoskeleton with membrane proteins. Red dashed circles emphasize such examples that
may be measured in vivo. F, force.
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that are enhanced by mechanical force due to
allosteric regulation. J. Biol. Chem. 283,
11596–11605.
10. del Rio, A., Perez-Jimenez, R., Liu, R.,
Roca-Cusachs, P., Fernandez, J.M., and
Sheetz, M.P. (2009). Stretching single talin
rod molecules activates vinculin binding.
Science 323, 638–641.
11. Greenleaf, W.J., Woodside, M.T., and
Block, S.M. (2007). High-resolution,
single-molecule measurements of biomolecular
motion. Annu. Rev. Biophys. Biomol. Struct. 36,
171–190.
12. Bustamante, C., Cheng, W., and Meija, Y.X.
(2011). Revisiting the central dogma one
molecule at a time. Cell 144, 480–497.
13. Glynn, S.E., Martin, A., Nager, A.R., Baker, T.A.,
and Sauer, R.T. (2009). Structures of
asymmetric ClpX hexamers reveal
nucleotide-dependent motions in a AAA+
protein-unfolding machine. Cell 139, 744–756.
14. Dufre ˆne, Y.F., Evans, E., Engel, A., Helenius, J.,
Gaub, H.E., and Mu ¨ller, D.J. (2011). Five
challenges to bringing single-molecule force
spectroscopy into living cells. Nat. Methods 8,
123–127.
1Institute of Condensed Matter and
Nanosciences, Universite ´ catholique de
Louvain, Croix du Sud 2/18, B-1348
Louvain-la-Neuve, Belgium.2ETH Zu ¨rich,
Department of Biosystems Science and
Engineering, 4058 Basel, Switzerland.
E-mail: Yves.Dufrene@uclouvain.be,
daniel.mueller@bsse.ethz.ch
DOI: 10.1016/j.cub.2011.04.046
Epithelial Organization: New
Perspective on a-Catenin from an
Ancient Source
Cadherins and catenins evidently partnered at the dawn of the animal kingdom
to enable the first polarized epithelium, and perhaps animal evolution itself.
New evidence from a primitive slime mold, however, suggests that a- and
b-catenins may have engaged this function independently, long before
cadherins arrived on the scene.
Albert B. Reynolds
Life as we know it evolved from
a common single-celled ancestor.
While the origin of life remains
shrouded in mystery, the history of life
is knowable and encoded in the
genomes of currently living organisms.
Through whole genome sequencing of
diverse life forms, scientists are
engaged in a high-tech journey of
molecular time travel. As new species
evolve, their predecessors are not lost,
but instead coexist over time as
separate species and coincidentally
as living molecular records of each
ancestral genome. These records
therefore hold the secrets to our past
in the form of a continuous thread of
molecular relationships that connect
all species inevitably to the ultimate
ancestral genome (Figure 1A).
Molecular time travel, however, is
essentially genome hopping, and
therein lies the rub: travel is limited to
sequenced genomes and, relatively
speaking, there are not all that many to
choose from. Nonetheless, the records
are there for the taking and scientists
can cherry-pick the most interesting
historical events by identifying relevant
organisms for genome sequencing
and/or analysis.
One such event revolves around the
question of how single-celled
organisms managed the highly
improbable transition to multicellularity
that led ultimately to metazoan
evolution and the animal kingdom.
Over several billion years, it appears
that multicellularity arose
independently by several mechanisms,
giving rise to animals, plants, and fungi
[1]. For animals, multicellularity has
been closely associated with the
arrival of cadherin-mediated cell–cell
adhesion [2]. Indeed, cadherins are
virtually absent from all other forms
of multicellular life, including
plants, fungi and slime molds.
The first cadherins on record appear
in the presumed single-celled
ancestors of metazoans, the
choanoflagellates — unicellular
colony-forming flagellates that swim
and prey on bacteria. Interestingly,
the feeding cells lining the oral cavity
of sponges, the first true animals, are
called choanocytes because they look
and feed like choanoflagellates.
However, the cadherins found in
choanoflagellates lack the cytoplasmic
catenin-binding domain found in
metazoan ‘classical’ cadherins, and
catenins are missing altogether. Thus,
classical cadherins and catenins came
together for the first time in the sponge,
the most primitive metazoan, evidently
enabling the organization of the first
complex epithelium and patterns of
embryogenesis that separate animals
from all other complex forms of life [3].
The extraordinary success of the
cadherin–catenin complex is
evidenced by repeated duplication
and diversification of the classical
cadherins in vertebrates to more than
26 members, most of which use the
same basic set of p120-, a- and
b-catenin building blocks. The core
design has thus been recycled for
half a billion years while simultaneously
serving as a key driver of vertebrate
cell- and tissue-diversification.
The scenario is a virtual indictment
of cadherins and catenins as partners
in crime and co-dependent enablers
of epithelial polarity and tissue
differentiation, if not animal evolution
itself.
However, a recent study by
Dickinson and colleagues [4],
published in Science, now reports
that ancient a- and b-catenins promote
epithelial organization in a primitive
slime mold. These findings are highly
unexpected on the grounds that
slime molds predate metazoans, do
not make cadherins and should not
be in the business of organizing
epithelia with catenins. The authors
identify a previously unappreciated
a-catenin-like protein (Dda-catenin) in
the social amoeba Dictyostelium
discoidium, also classified as a slime
mold. Next, they establish functional
analogy to murine a-catenin by
demonstrating a direct interaction
between Dda-catenin and Aardvark
(the Dictyostelium homologue of
b-catenin), and between Dda-catenin
and murine b-catenin. Thus, it appears
that Dictyostelium encodes primitive
forms of a- and b-catenin that interact
with one another and presumably
function together.
Dickinson et al. [4] go on to identify
andfunctionallycharacterizeaprimitive
polarized epithelium referred to as the
‘tipepithelium’becauseitformsaround
the tip of a spore structure, or fruiting
body,duringamulticellularphaseofthe
Dictyostelium life cycle. Interestingly,
they show that Dda-catenin promotes
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