Directed

evolution

of

a

biterminal

bacterial

display

scaffold

enhances

the

display

of

diverse

peptides

Bacterial

cell-surface

display

systems

coupled

with

quantitative

screening

methods

offer

the

potential

to

expand

protein

engineering

capabilities

To

more

fully

exploit

this

potential

a

unique

bacterial

surface

display

scaffold

was

engineered

to

display

peptides

more

efficiently

from

the

surface

exposed

C

and

N-termini

of

a

circularly

permuted

outer

membrane

protein

Using

directed

evolution

efficient

membrane

localization

of

a

circularly

permuted

OmpX

-LRB-

CPX

-RRB-

display

scaffold

was

rescued

thereby

improving

the

presentation

of

diverse

passenger

peptides

on

the

cell

surface

Random

and

targeted

mutagenesis

directed

towards

linkers

joining

the

native

N

and

C-termini

of

OmpX

coupled

with

screening

by

FACS

yielded

an

enhanced

CPX

-LRB-

eCPX

-RRB-

variant

which

localized

to

the

outer

membrane

as

efficiently

as

the

non-permuted

parent

Interestingly

enhancing

substitutions

coincided

with

a

C-terminal

motif

conserved

in

outer

membrane

proteins

Surface

localization

of

various

passenger

peptides

and

mini-proteins

was

expedited

using

eCPX

relative

to

that

achieved

with

the

parent

scaffold

The

new

variant

also

permitted

simultaneous

display

and

labeling

of

distinct

peptides

on

structurally

adjacent

C

and

N-termini

thus

enabling

display

level

normalization

during

library

screening

and

the

display

of

bidentate

or

dimeric

peptides

Consequently

the

evolved

scaffold

eCPX

expands

the

range

of

applications

for

bacterial

display

Finally

this

approach

provides

a

route

to

improve

the

performance

of

cell-surface

display

vectors

for

protein

engineering

and

design

IntroductionDisplay

methodologies

have

proven

invaluable

to

the

discovery

production

and

optimization

of

proteins

and

peptides

in

a

variety

of

biotechnological

applications

Various

approaches

including

phage

display

-LRB-

Smith

-RRB-

mRNA

-LRB-

Wilson

et

al.

-RRB-

and

DNA

display

-LRB-

Yonezawa

et

al.

-RRB-

ribosome

display

-LRB-

Mattheakis

et

al.

Hanes

and

Pluckthun

-RRB-

eukaryotic

virus

display

-LRB-

Muller

et

al.

Bupp

and

Roth

-RRB-

yeast

display

-LRB-

Boder

and

Wittrup

-RRB-

and

bacterial

display

-LRB-

Lu

et

al.

-RRB-

have

been

developed

to

screen

diverse

molecular

repertoires

for

desired

activities

In

particular

bacterial

display

libraries

have

enabled

antibody

affinity

maturation

-LRB-

Daugherty

et

al.

-RRB-

the

discovery

of

protein-binding

peptides

-LRB-

Bessette

et

al.

-RRB-

the

isolation

of

cell-specific

ligands

-LRB-

Nakajima

et

al.

Dane

et

al.

-RRB-

and

the

identification

of

optimal

protease

substrates

-LRB-

Boulware

and

Daugherty

-RRB-

One

of

the

key

advantages

of

bacterial

surface

display

is

the

ability

to

use

flow

cytometry

for

quantitative

screening

of

the

libraries

allowing

for

real-time

analysis

of

binding

affinity

and

specificity

to

optimize

the

screening

process

-LRB-

Wittrup

-RRB-

Additionally

ease

of

genetic

manipulation

high

transformation

efficiency

and

rapid

growth

rate

make

Escherichia

coli

a

well-suited

host

for

display

A

broad

range

of

bacterial

surface

display

systems

have

been

developed

allowing

for

insertional

or

terminally

fused

peptides

and

proteins

to

be

displayed

on

the

cell

surface

Several

outer

membrane

proteins

and

cellular

appendage

proteins

have

been

used

to

present

polypeptides

as

insertional

fusions

-LRB-

Charbit

et

al.

Taschner

et

al.

Bessette

et

al.

-RRB-

Ice

nucleation

protein

-LRB-

Jung

et

al.

-RRB-

intimins

-LRB-

Christmann

et

al.

-RRB-

and

LppOmpA

-LRB-

Francisco

et

al.

-RRB-

have

been

used

to

display

proteins

on

the

C-terminus

of

a

transmembrane

scaffold

while

N-terminal

display

has

been

accomplished

using

autotransporters

protease

-LRB-

Maurer

et

al.

-RRB-

and

EstA

-LRB-

Becker

et

al.

-RRB-

Recently

a

unique

bacterial

display

scaffold

was

developed

that

allows

for

both

N

and

C-terminal

display

from

a

circularly

permuted

variant

of

outer

membrane

protein

OmpX

-LRB-

CPX

-RRB-

-LRB-

Rice

et

al.

-RRB-

This

scaffold

enabled

display

of

peptides

on

both

termini

but

with

reduced

efficiency

when

compared

to

that

obtained

using

insertions

into

OmpX

Reduced

membrane

localization

of

CPX

may

result

from

slower

folding

rates

and

reduced

stability

that

has

been

described

previously

for

circularly

permuted

proteins

-LRB-

Heinemann

and

Hahn

-RRB-

Regardless

reduced

display

efficiency

makes

it

necessary

to

employ

longer

induction

times

to

achieve

sufficient

display

for

screening

via

fluorescence-activated

cell

sorting

-LRB-

FACS

-RRB-

Importantly

inefficient

display

can

also

create

an

undesired

selection

pressure

resulting

in

growth

biases

reduced

viability

or

differing

levels

of

passenger

localization

on

the

cell

surface

As

a

result

library

screening

based

on

cell

fluorescence

can

favor

passengers

most

efficiently

localized

to

the

surface

rather

than

passengers

improved

for

the

properties

of

interest

-LRB-

e.g.

binding

affinity

-RRB-

A

variety

of

proteins

have

been

circularly

permuted

to

create

novel

proteins

of

desired

topologies

For

instance

Guntas

et

al.

coupled

a

circularly

permuted

β-lactamase

with

maltose

binding

protein

to

create

a

switch

in

which

β-lactam

hydrolysis

activity

is

increased

in

the

presence

of

maltose

-LRB-

Guntas

et

al.

-RRB-

Also

fluorescent

proteins

have

been

circularly

permuted

to

create

molecular

sensors

that

respond

to

-LRB-

Baird

et

al.

-RRB-

In

these

instances

the

point

of

circular

permutation

can

have

important

consequences

for

protein

function

Random

circular

permutations

often

yield

inactive

or

unfolded

variants

-LRB-

Graf

and

Schachman

-RRB-

and

those

variants

retaining

activity

often

fold

more

slowly

and

are

less

stable

than

the

non-permuted

parent

-LRB-

Lindqvist

and

Schneider

-RRB-

With

the

aim

of

rescuing

the

reduced

surface

localization

of

CPX

relative

to

the

non-permuted

OmpX

we

used

targeted

combinatorial

mutagenesis

and

directed

evolution

to

identify

CPX

variants

that

would

localize

passenger

peptides

to

the

cell

surface

with

an

efficiency

equivalent

to

that

of

native

OmpX

This

strategy

yielded

a

more

robust

display

scaffold

to

present

diverse

peptides

as

C

and

N-terminal

fusions

on

the

surface

of

bacteria

ResultsCircularly

permuted

OmpX

librariesWe

previously

reported

a

new

type

of

protein

scaffold

for

polypeptide

display

that

allows

for

both

N

and

C-termini

to

be

exterior

to

the

cell

using

circularly

permuted

outer

membrane

protein

OmpX

-LRB-

CPX

-RRB-

-LRB-

Rice

et

al.

-RRB-

The

CPX

protein

scaffold

consists

of

the

native

OmpX

signal

sequence

which

is

cleaved

after

translocation

a

sequence

with

an

embedded

SfiI

restriction

site

-LRB-

GQSGQ

-RRB-

after

which

peptides

may

be

inserted

a

flexible

linking

sequence

-LRB-

GGQSGQ

-RRB-

amino

acids

of

the

mature

OmpX

a

GGSG

linker

joining

the

native

C

and

N-termini

and

finally

amino

acids

of

the

mature

OmpX

To

assess

the

extent

of

display

a

disulfide-constrained

streptavidin-binding

peptide

-LRB-

SApep

-RRB-

with

the

amino

acid

sequence

AECHPQGPPCIEGRK

-LRB-

Giebel

et

al.

-RRB-

was

fused

to

the

N-terminus

of

CPX

allowing

cell

labeling

using

a

fluorescently

conjugated

streptavidin

probe

and

measurement

of

display

levels

using

cytometry

CPX

yielded

a

reduced

level

of

peptide

display

when

compared

to

cells

displaying

the

same

peptide

presented

as

an

insertion

within

the

corresponding

region

of

OmpX

-LRB-

Fig.

-RRB-

The

reduced

display

ability

requires

longer

induction

periods

to

allow

for

sufficient

fluorescence

labeling

and

library

screening

thereby

causing

cell

stress

that

can

result

in

growth

biases

during

library

screening

-LRB-

Daugherty

et

al.

-RRB-

Fig.

of

a

streptavidin-binding

peptide

using

OmpX

CPX

and

eCPX

Flow

cytometric

analysis

of

E.

coli

displaying

a

streptavidin-binding

peptide

-LRB-

AECHPQGPPCIEGRK

-RRB-

-LRB-

Giebel

et

al.

-RRB-

as

an

insertion

into

OmpX

an

N-terminal

fusion

to

CPX

or

an

N-terminal

fusion

to

eCPX

after

varying

durations

of

induction

The

cells

were

induced

at

room

temperature

for

time

increments

between

and

min

then

labeled

with

nM

SA

PE.The

CPX

scaffold

was

constructed

using

an

arbitrarily

chosen

flexible

linker

-LRB-

GGSG

-RRB-

to

join

the

native

N

and

C-termini

Thus

we

reasoned

that

alternative

linkers

and

point

mutations

within

CPX

could

enhance

the

display

of

peptides

on

the

cell

surface

To

improve

the

display

characteristics

of

CPX

various

regions

of

the

transmembrane

protein

were

targeted

for

mutagenesis

An

optimized

linker

region

joining

the

native

N

and

C-termini

was

identified

by

generating

and

screening

four

libraries

allowing

for

three

-LRB-

-RRB-

four

-LRB-

-RRB-

five

-LRB-

-RRB-

and

six

-LRB-

-RRB-

random

residues

to

be

inserted

in

place

of

the

GGSG

linker

using

the

degenerate

codon

NNK

Each

library

was

screened

separately

using

FACS

for

clones

exhibiting

a

high

level

of

fluorescence

after

min

of

induction

indicating

increased

display

of

SApep

binding

to

streptavidin

R-phycoerythrin

-LRB-

SA

PE

-RRB-

Under

these

conditions

the

parent

CPX

scaffold

yielded

display

levels

only

slightly

greater

than

background

autofluorescence

making

the

selection

of

improved

mutants

efficient

After

sorting

the

display

level

of

several

clones

was

measured

using

cytometry

and

their

sequences

were

determined

by

DNA

sequencing

-LRB-

Table

I

-RRB-

Isolated

clones

exhibited

to

improved

display

compared

to

CPX

after

only

a

min

induction

period

The

identified

linker

sequences

exhibited

a

preference

for

basic

residues

and

glycine

was

present

at

the

first

position

of

the

linker

in

of

clones

characterized

In

addition

four

of

the

clones

had

unintended

mutations

preceding

the

native

C-terminus

two

with

the

substitution

and

two

others

with

Interestingly

these

four

clones

were

among

the

most

improved

display

scaffolds

isolated

The

average

display

level

of

the

selected

clones

from

each

library

increased

with

increasing

linker

length

and

was

highest

for

and

linker

clones.Table

IMutations

improving

CPX

surface

localizationClonePositionsLinkerRelative

display

bThree

residue

linker

library

a

a

a

residue

linker

library

residue

linker

library

residue

linker

library

generation

library

fluorescence

above

background

after

min

of

expression

bfold

fluorescence

above

background

after

min

of

expression.In

an

attempt

to

further

enhance

peptide

display

the

amino

acid

linker

that

joined

the

passenger

peptide

to

the

N-terminus

of

the

display

scaffold

was

also

targeted

for

mutagenesis

and

screening

for

improved

variants

A

library

was

created

in

place

of

the

original

linking

sequence

GGQSGQ

by

randomizing

these

six

residues

Screening

yielded

four

clones

exhibiting

a

improved

display

when

compared

with

CPX

Sequencing

did

not

reveal

a

consensus

within

the

target

linker

region

Instead

clones

with

enhanced

display

possessed

a

non-targeted

substitution

of

either

or

Since

the

randomly

selected

library

members

from

the

initial

pool

did

not

possess

mutations

outside

of

the

intended

region

these

advantageous

substitutions

were

rare

and

likely

arose

from

PCR

errors

In

parallel

a

library

was

generated

with

random

residues

at

the

surface

exposed

C-terminus

of

CPX

since

native

outer

membrane

proteins

-LRB-

Omps

-RRB-

possess

a

conserved

C-terminal

motif

thought

to

aid

in

Omp

membrane

insertion

or

assembly

-LRB-

Bos

and

Tommassen

-RRB-

Four

clones

were

isolated

from

this

library

that

exhibited

improved

peptide

display

Again

these

variants

did

not

share

consensus

in

the

randomized

region

but

each

carried

the

spontaneous

mutation

These

results

suggest

that

the

amino

acid

compositions

of

the

new

termini

derived

from

circular

permutation

have

little

effect

on

the

rate

of

assembly

and

display

of

CPX

whereas

residues

and

play

a

key

role

in

proper

translocation

and

insertion

of

the

protein.A

final

library

was

designed

to

combine

the

display-enhancing

mutations

identified

within

the

most

improved

clones

from

the

first

generation

libraries

A

six

residue

linker

library

was

chosen

to

identify

optimal

linkers

to

connect

the

native

N

and

C-termini

since

longer

linkers

appeared

to

improve

display

-LRB-

Table

I

-RRB-

After

min

of

induction

individual

library

members

exhibited

higher

display

levels

on

average

when

compared

with

clones

with

five

amino

acid

linkers

-LRB-

Table

I

-RRB-

The

first

amino

acid

of

the

linker

was

fixed

to

glycine

since

it

was

highly

conserved

in

the

isolated

clones

The

third

and

sixth

positions

were

restricted

to

R/K/S

H/Q/N

using

the

codon

MRM

given

the

increased

frequency

of

these

residues

at

the

proposed

positions

in

clones

with

improved

function

and

the

remaining

three

positions

were

fully

randomized

Positions

and

where

beneficial

substitutions

were

observed

were

also

fully

randomized

Improved

variants

were

identified

using

two

rounds

of

MACS

followed

by

two

rounds

of

FACS

sorting

clones

exhibiting

the

highest

display

of

the

SApep

after

min

of

induction

Ten

clones

were

isolated

after

the

final

FACS

screening

-LRB-

Table

I

-RRB-

All

variant

scaffolds

identified

possessed

a

more

bulky

hydrophobic

residue

-LRB-

I/L/V

F

-RRB-

in

place

of

alanine

at

position

a

consensus

for

serine

at

position

and

a

high

frequency

of

basic

residues

Arg

and

Lys

within

the

linking

region

The

display

enhancing

substitutions

and

are

located

immediately

upstream

of

the

native

C-terminus

of

OmpX

-LRB-

Fig.

-RRB-

Fig.

of

enhancing

mutations

within

eCPX

Image

was

created

using

Molecular

Operating

Environment

using

the

reported

structure

of

OmpX

-LRB-

Vogt

and

Schulz

-RRB-

joining

the

N

and

C-termini

with

the

six

residue

linker

GSKSRR

and

creating

the

new

termini

within

the

second

extracellular

loop

Enhancing

substitutions

and

are

shown

as

space

filling

residues.Expression

characteristics

of

optimized

CPXThe

scaffold

variant

exhibiting

the

most

improved

display

characteristics

or

enhanced

CPX

-LRB-

eCPX

-RRB-

was

then

compared

to

parental

OmpX

and

CPX

scaffolds

The

cell-surface

display

level

of

SApep

was

measured

at

incremental

times

after

induction

of

expression

for

these

three

scaffolds

This

peptide

was

displayed

either

at

the

N-terminus

-LRB-

CPX

and

eCPX

-RRB-

or

as

an

insertional

fusion

within

the

second

extracellular

loop

of

OmpX

The

level

of

display

was

measured

before

and

after

and

min

of

induction

using

flow

cytometry

-LRB-

Fig.

-RRB-

The

display

rate

of

eCPX

was

substantially

improved

relative

to

that

of

CPX

and

even

slightly

higher

than

that

of

OmpX

After

only

min

of

expression

the

level

of

display

of

eCPX-nSApep

was

above

background

autofluorescence

Introducing

and

into

OmpX

resulted

in

nearly

identical

display

of

SApep

relative

to

that

obtained

with

OmpX

-LRB-

data

not

shown

-RRB-

To

determine

whether

the

enhanced

display

using

the

eCPX

scaffold

was

a

general

effect

or

specific

to

the

streptavidin

binding

peptide

SApep

several

unrelated

passenger

peptides

were

fused

to

the

N-terminus

of

CPX

and

eCPX

and

their

display

levels

were

compared

-LRB-

Fig.

-RRB-

Surprisingly

a

disulfide-constrained

peptide

binding

to

C-reactive

protein

-LRB-

CRPpep

-RRB-

-LRB-

EWACNDRGFNCQLQR

-RRB-

identified

by

bacterial

display

-LRB-

Bessette

et

al.

-RRB-

displayed

with

eCPX

yielded

nearly

higher

florescence

labeling

than

that

for

CPX

after

only

min

of

expression

In

fact

the

fluorescence

of

cells

displaying

CRPpep

from

CPX

could

not

be

distinguished

from

background

-LRB-

Fig.

-RRB-

Similarly

the

tag

epitope

-LRB-

-RRB-

-LRB-

MASMTGGQQMG

-RRB-

was

displayed

more

efficiently

by

eCPX

than

by

CPX

A

disulfide-constrained

peptide

binding

to

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

identified

previously

using

phage

display

-LRB-

Fairbrother

et

al.

-RRB-

was

also

displayed

over

more

efficiently

within

eCPX

Additionally

an

IgG

binding

mini-protein

-LSB-

a

minimized

version

of

the

Z-domain

from

protein

A

-LRB-

Braisted

and

Wells

-RRB-

-RSB-

composed

of

acids

that

form

two

antiparallel

α-helices

exhibited

a

display

level

roughly

higher

than

that

from

CPX

after

a

h

induction

period

Finally

a

proline-rich

peptide

-LRB-

PAPSIDRSTKPPL

-RRB-

known

to

bind

to

the

C-terminal

domain

of

Mona

-LRB-

Harkiolaki

et

al.

-RRB-

was

expressed

as

a

C-terminal

fusion

using

both

CPX

and

eCPX

Similar

to

the

improvements

of

display

at

the

N-terminus

the

display

level

after

only

min

of

expression

of

using

eCPX

was

improved

by

compared

to

display

with

CPX

Thus

for

all

peptides

investigated

the

eCPX

scaffold

increased

display

levels

when

compared

to

the

parental

CPX.Fig

of

various

peptides

and

mini-proteins

using

eCPX

-LRB-

shaded

-RRB-

and

CPX

-LRB-

white

-RRB-

as

measured

using

flow

cytometry

The

X-axis

indicates

the

fold

fluorescence

above

background

for

each

protein

target

in

the

corresponding

fluorescent

channel

was

labeled

with

Mona

which

is

fused

to

the

fluorescent

protein

YPet

CRPpep

and

were

labeled

with

biotinylated

CRP

and

VEGF

respectively

then

labeled

with

SA

PE

Mini-Z

and

were

labeled

with

conjugated

human

IgG

and

tag

monoclonal

IgG

respectively

SApep

was

detected

with

SA

PE.Since

the

increased

expression

of

the

scaffold

could

potentially

result

in

a

reduction

in

cell

viability

the

viability

of

E.

coli

populations

displaying

two

different

peptides

was

measured

as

a

function

of

the

duration

of

induction

of

expression

The

IgG

binding

mini-Z

helix

was

selected

since

it

exhibited

reduced

display

in

the

improved

eCPX

scaffold

Viability

was

measured

at

and

min

after

induction

using

CFU

assays

and

compared

to

that

for

parental

strain

-LRB-

-RRB-

lacking

an

expression

cassette

Under

ordinary

display

conditions

-LRB-

i.e.

induction

for

less

than

min

-RRB-

a

measurable

loss

in

viability

could

not

be

observed

After

prolonged

induction

-LRB-

min

-RRB-

cells

expressing

the

IgG

binding

minihelix

-LRB-

miniZ

-RRB-

exhibited

a

loss

of

viability

-LRB-

data

not

shown

-RRB-

while

cells

displaying

SApep

exhibited

viability

indistinguishable

from

that

of

cells

that

do

not

express

the

scaffold

-LRB-

-RRB-

Biterminal

display

using

eCPXTwo

distinct

peptides

were

simultaneously

displayed

on

the

structurally

adjacent

N

and

C-termini

of

eCPX

SApep

was

fused

to

the

N-terminus

and

the

peptide

fused

to

the

C-terminus

-LRB-

-RRB-

Labeling

with

fluorescent

probes

SA

PE

-LRB-

red

-RRB-

and

YPet-Mona

-LRB-

Nguyen

and

Daugherty

-RRB-

-LRB-

green

-RRB-

enabled

independent

detection

of

each

peptide

using

flow

cytometry

To

determine

the

ability

of

eCPX

to

simultaneously

display

these

two

peptides

cells

expressing

the

biterminal

display

scaffold

were

labeled

with

SA

PE

only

YPet-Mona

only

or

both

probes

concurrently

If

the

peptides

bound

to

their

respective

receptors

independently

-LRB-

i.e.

without

any

steric

clashes

-RRB-

there

should

be

no

difference

between

the

extent

of

single

color

labeling

-LRB-

fluorescence

intensity

-RRB-

of

the

sample

labeled

with

one

probe

and

that

labeled

with

both

fluorescent

probes

simultaneously

However

simultaneous

labeling

of

cells

expressing

a

fusion

protein

of

the

form

with

SA

PE

and

YPet-Mona

or

with

each

probe

separately

yielded

differing

extents

of

labeling

consistent

with

steric

interference

between

these

two

large

fluorescent

probes

-LRB-

and

kDa

respectively

-RRB-

Specifically

the

fluorescence

of

the

cells

when

labeled

with

only

one

probe

was

always

greater

than

the

fluorescence

in

the

corresponding

channel

of

the

cells

when

labeled

with

both

probes

simultaneously

In

an

attempt

to

reduce

steric

interference

a

long

flexible

linker

of

the

form

-LRB-

GGGS

-RRB-

was

inserted

between

SApep

and

eCPX

resulting

in

a

total

linker

length

of

amino

acids

causing

SApep

to

be

further

from

the

cell

surface

and

thus

increasing

the

distance

between

the

two

peptides

Using

this

long

linker

independent

labeling

of

each

displayed

peptide

was

improved

-LRB-

Fig.

-RRB-

These

results

indicate

that

the

use

of

a

long

unstructured

linker

can

increase

the

accessibility

of

large

proteins

to

peptides

simultaneously

displayed

at

both

termini

of

eCPX

without

substantially

reducing

the

level

of

display.Fig

cytometric

measurement

of

simultaneous

N

and

C-terminal

display

-LRB-

bi-terminal

display

-RRB-

Overlays

of

two-parameter

histograms

resulting

from

clones

displaying

SApep

and

on

the

N

and

C-terminus

respectively

with

a

-LRB-

A

-RRB-

or

a

-LRB-

B

-RRB-

residue

linker

between

SApep

and

the

N-terminus

of

eCPX

In

both

-LRB-

A

-RRB-

and

-LRB-

B

-RRB-

plots

the

four

distinct

populations

consist

of

non-displaying

cells

mock-labeled

with

SA

PE

and

Ypet-Mona

-LRB-

bottom

left

population

-RRB-

cells

that

display

SApep

and

labeled

with

only

SA

PE

-LRB-

top

left

population

-RRB-

with

SA

PE

and

YPet-Mona

-LRB-

top

right

population

-RRB-

or

with

only

YPet-Mona

-LRB-

bottom

right

population

-RRB-

DiscussionThe

application

of

bacterial

display

technology

to

a

broader

range

of

protein

engineering

applications

has

been

hindered

by

the

absence

of

robust

validated

display

scaffolds

We

recently

reported

a

circularly

permuted

outer

membrane

protein

X

-LRB-

CPX

-RRB-

having

the

unique

characteristic

that

both

C

and

N-termini

of

the

scaffold

become

localized

on

the

bacterial

cell

surface

-LRB-

Rice

et

al.

-RRB-

This

biterminal

scaffold

provides

a

means

for

expression

normalization

to

improve

affinity

screening

analogous

to

yeast

display

-LRB-

Boder

and

Wittrup

-RRB-

as

well

as

a

means

to

display

dimeric

peptides

However

the

permuted

scaffold

exhibited

reduced

surface

localization

efficiency

precluding

presentation

of

large

peptides

or

the

display

of

two

unique

peptides

simultaneously

from

structurally

adjacent

termini

Here

semi-rational

design

and

directed

evolution

were

used

to

enhance

the

surface

localization

efficiency

of

CPX

to

improve

the

display

of

diverse

peptides

To

identify

CPX

scaffold

variants

with

optimal

linker

sequences

for

joining

the

native

C

and

N-termini

four

separate

libraries

with

three

four

five

or

six

random

linker

amino

acids

were

screened

Improved

scaffold

variants

revealed

a

preference

for

longer

linkers

of

five

to

six

residues

a

strict

consensus

for

glycine

at

the

first

position

of

the

linker

and

a

high

frequency

of

basic

residues

in

the

remaining

positions

Unintended

substitutions

-LRB-

-RRB-

near

the

native

C-terminus

of

OmpX

greatly

increased

the

display

level

These

substitutions

probably

arose

from

rare

errors

introduced

during

PCR

which

were

enriched

from

the

large

libraries

-LRB-

-RRB-

After

directed

evolution

the

final

variant

with

the

most

improved

display

-LRB-

eCPX

-RRB-

carried

substitutions

and

with

a

linker

sequence

of

GSKSRR.Two

substitutions

-LRB-

-RRB-

identified

to

improve

surface

localization

occur

at

the

native

C-terminus

of

OmpX

and

have

a

high

degree

of

similarity

to

the

C-termini

of

outer

membrane

proteins

that

lack

periplasmic

domains

-LRB-

Table

II

-RRB-

even

though

these

Omps

share

little

identity

These

results

support

previous

observations

that

the

C-terminal

sequence

serves

an

important

role

in

membrane

assembly

and/or

translocation

-LRB-

Struyve

et

al.

-RRB-

The

beneficial

eCPX

substitutions

discovered

could

potentially

enhance

the

ability

of

chaperones

to

recognize

and

assemble

this

topological

variant

Interestingly

the

extended

C-terminal

motif

resulting

from

directed

evolution

-LRB-

L/VXYRFG

-RRB-

is

conserved

in

OmpA

homologs

which

utilize

this

motif

in

their

last

transmembrane

β-strand

that

precedes

a

C-terminal

periplasmic

domain

-LRB-

Table

II

-RRB-

Our

results

suggest

that

glycine

is

required

to

allow

the

internal

as

opposed

to

terminal

YRF

motif

to

interact

with

a

putative

chaperone

The

improved

linkers

also

include

an

increased

frequency

of

charged

residues

that

could

serve

to

increase

solubility

and

decrease

aggregation

to

allow

more

protein

to

insert

into

the

membrane.Table

IIComparison

of

bacterial

outer

membrane

proteins

C-terminal

sequences

with

eCPXOmp

IDC-terminal

sequenceaeCPXDVGTWILSVGYRFGSKSRRAT

of

last

membrane

β-strand

of

OmpA

between

eCPX

-LRB-

with

substitutions

-RRB-

and

other

bacterial

outer

membrane

protein

C-termini

bOmpA

homologues

with

a

periplasmic

domain

following

the

transmembrane

strand

exhibit

a

highly

conserved

glycine

residue

following

the

C-terminal

YRF

motif

of

transmembrane

proteins.The

evolution

of

an

efficiently

localized

biterminal

display

scaffold

-LRB-

eCPX

-RRB-

provides

several

important

new

capabilities

for

peptide

engineering

First

eCPX

increased

the

display

rate

of

various

unrelated

peptide

fusions

on

either

the

N

or

C-terminus

when

compared

to

that

achieved

using

the

parental

CPX

Increased

display

improves

library

screening

efficiency

using

FACS

and

promotes

the

recovery

of

peptides

that

are

otherwise

difficult

to

display

For

example

the

display

of

CRPpep

using

CPX

was

only

marginally

detectable

after

min

of

expression

but

the

same

peptide

displayed

using

eCPX

provided

a

fluorescent

signal

above

background

If

a

peptide

library

displayed

on

CPX

was

screened

for

CRP

binding

peptides

this

sequence

and

others

like

it

may

not

have

been

isolated

due

to

the

difficulties

in

presenting

this

peptide

using

CPX

Though

it

should

be

noted

that

excessive

display

levels

above

that

required

for

screening

by

FACS

should

be

avoided

to

minimize

avidity

interactions

with

target

proteins

In

fact

using

eCPX

induction

periods

as

short

as

min

can

be

sufficient

for

affinity

screening

of

peptide

libraries

-LRB-

unpublished

observations

-RRB-

Importantly

quantifying

the

display

level

during

library

screening

by

labeling

of

a

C-terminal

peptide

such

as

allows

fine

affinity

discrimination

-LRB-

VanAntwerp

and

Wittrup

-RRB-

that

is

peptides

with

a

high

affinity

but

low

display

level

can

be

differentiated

from

peptides

with

a

high

display

but

moderate

affinity

Moreover

efficient

biterminal

display

allows

for

the

potential

to

generate

peptide

libraries

on

each

terminus

where

both

peptides

can

bind

to

distinct

epitopes

of

a

target

protein

resulting

in

increased

binding

affinity

and

specificity

through

avidity

Finally

C-terminal

display

with

eCPX

should

enable

new

applications

such

as

screening

for

preferred

PDZ

C-terminal

binding

motifs

-LRB-

Harris

and

Lim

-RRB-

Engineering

of

the

bacterial

display

scaffold

eCPX

yielded

a

circularly

permuted

transmembrane

protein

that

has

both

termini

presented

on

the

exterior

of

the

cell

and

inserts

into

the

outer

membrane

as

efficiently

as

the

non-permuted

parent

The

amino

acid

sequence

used

to

join

the

termini

played

a

major

role

in

the

proper

function

of

CPX

and

two

substitutions

adjacent

to

the

native

C-terminus

aided

in

display

of

peptides

using

eCPX

This

novel

protein

scaffold

substantially

improves

the

robustness

of

bacterial

surface

display

via

CPX

and

when

combined

with

modern

cell

sorting

instrumentation

creates

new

opportunities

for

peptide

engineering

More

generally

similar

linker

mutagenesis

strategies

coupled

with

efficient

screening

methods

should

prove

useful

for

optimizing

other

display

scaffolds

and

circularly

permuted

proteins

Materials

and

methodsBacterial

strains

reagents

and

plasmidsAll

experiments

were

performed

with

E.coli

strain

-LRB-

F

Δ

-LRB-

ara-leu

-RRB-

Δ

-LRB-

lac

-RRB-

rpsL

-LRB-

StrR

-RRB-

-LRB-

rK

mK

-RRB-

mcrA

-RRB-

-LRB-

Casadaban

and

Cohen

-RRB-

All

plasmid

constructs

utilize

-LRB-

Cmr

-RRB-

-LRB-

Guzman

et

al.

-RRB-

with

the

promoter

araBAD

operon

and

the

origin

of

replication

-LRB-

low-copy

number

-RRB-

KOD

Hot

Start

DNA

Polymerase

-LRB-

Novagen

-RRB-

was

used

for

PCRs

Primers

were

from

Operon

restriction

enzymes

from

New

England

BioLabs

SA

PE

from

Molecular

Probes

and

streptavidin-coated

magnetic

microbeads

-LRB-

MyOne

Streptavidin

-RRB-

were

from

Invitrogen

Qiagen

mini-preps

and

gel

extraction

kits

were

used

for

DNA

preparation

Ni-NTA

agarose

for

protein

purification

was

from

Qiagen

and

B-PER

II

bacterial

protein

extraction

reagent

was

from

Pierce

Biotechnology.Vector

and

library

constructionConstruction

of

circularly

permutated

OmpX

-LRB-

CPX

-RRB-

was

described

previously

-LRB-

Rice

et

al.

-RRB-

To

monitor

the

display

level

a

streptavidin

binding

peptide

was

fused

to

the

N-terminus

of

CPX

as

described

in

earlier

work

this

plasmid

was

termed

To

generate

the

libraries

that

joined

the

original

N

and

C-termini

of

OmpX

with

three

to

six

random

residues

primers

-LRB-

see

Supplementary

data

available

at

PEDS

Online

-RRB-

were

used

-LRB-

respectively

-RRB-

as

the

reverse

primer

and

as

the

forward

primer

with

as

the

template

Primers

are

listed

in

the

supplementary

material

The

random

positions

were

encoded

using

NNK

codons

allowing

for

all

amino

acids

and

the

amber

stop

codon

The

product

of

the

PCR

reaction

was

gel

purified

then

used

as

a

forward

primer

for

the

next

reaction

using

as

a

reverse

primer

and

again

with

as

the

template

The

product

was

then

gel

purified

digested

with

SfiI

and

gel

purified

again

The

digested

insert

was

ligated

into

the

similarly

digested

vector

Ligation

products

were

desalted

and

electroporated

into

electro-competent

yielding

transformants

respectively

To

create

the

second

generation

library

-LRB-

CPX-directed

-RRB-

primers

and

were

used

to

randomize

positions

and

using

as

the

template

The

product

was

then

used

as

a

template

for

PCR

with

and

adding

the

second

generation

library

residues

The

primer

encoded

for

G

at

the

first

position

of

the

linker

and

used

a

restricted

codon

of

MRM

to

encode

for

residues

R

K

S

H

Q

or

N

at

positions

and

of

the

linker

the

remaining

positions

used

NNK

The

product

of

the

previous

PCR

reaction

was

then

used

as

a

forward

primer

for

the

next

reaction

using

as

a

reverse

primer

and

as

the

template

The

product

was

then

digested

and

ligated

into

the

similarly

digested

vector

Ligation

products

were

desalted

and

electroporated

into

electro-competent

yielding

transformants.The

various

binding

peptides

were

fused

to

the

N-terminus

of

CPX

and

eCPX

using

a

linker

of

GGQSGQS

PCR

was

used

with

as

the

template

as

the

reverse

primer

and

with

forward

primers

for

the

CRP

binding

peptide

-LRB-

EWACNDRGF.NCQLQR

-RRB-

or

for

the

VEGF

binding

peptide

-LRB-

VEPNCDIHVMWEWECFERL

-RRB-

The

products

were

digested

with

SfiI

and

ligated

into

similarly

digested

vector

and

electroporated

into

Primers

were

used

in

an

assembly

PCR

to

create

the

forward

primer

for

the

mini-Z-domain

-LRB-

FNMQQQRRFYEALHDPNLNEEQRNAKIKSIRDD

-RRB-

This

primer

with

and

template

was

used

in

PCR

The

product

was

digested

with

SfiI

and

ligated

into

similarly

digested

vector

and

electroporated

into

The

-LRB-

MASMTGGQQMG

-RRB-

was

created

using

overlap

extension

PCR

with

the

products

from

the

PCR

reaction

with

primers

and

The

products

were

digested

with

SfiI

and

ligated

into

similarly

digested

vector

and

electroporated

into

To

transfer

these

peptides

to

eCPX

the

vectors

containing

the

peptide

CPX

fusion

was

digested

with

PstI

and

KpnI

the

smaller

fragment

was

gel

extracted

and

ligated

to

the

similarly

digested

vector

of

eCPX

transferring

the

displayed

peptide

to

the

eCPX

plasmid

To

insert

the

peptide

-LRB-

PAPSIDRSTKPPL

-RRB-

at

the

C-terminus

of

CPX

and

eCPX

PCR

was

used

with

as

the

forward

primer

and

primers

as

the

reverse

primers

with

at

template

The

primers

also

encoded

a

linker

of

GGQSGQ

preceding

the

peptide

The

products

were

digested

with

SfiI

and

ligated

into

similarly

digested

vector

and

electroporated

into

The

gene

is

The

streptavidin

binding

peptide

was

removed

using

KpnI

and

HindIII

and

ligation

with

a

similarly

cut

insert

that

contained

no

N-terminally

fused

peptide

creating

To

insert

an

extended

linker

of

-LRB-

GGGS

-RRB-

between

the

streptavidin

binding

peptide

and

PCR

was

used

with

forward

primer

and

reverse

primers

with

as

template

The

product

was

then

gel

extracted

and

used

as

the

forward

primer

with

as

the

reverse

primer

and

as

template

The

product

was

gel

extracted

and

digested

with

SfiI

and

ligated

to

similarly

digested

vector

The

portion

after

the

OmpX

signal

sequence

was

now

GQSGQ

-LRB-

encoding

a

SfiI

site

-RRB-

AECHPQGPPCIEGRK

-LRB-

the

streptavidin

binding

peptide

-RRB-

-LRB-

GGGS

-RRB-

-LRB-

the

additional

linker

-RRB-

and

GGQSGQ

-LRB-

original

linker

-RRB-

followed

by

the

of

eCPX

with

the

peptide

on

the

C-terminus

the

gene

construct

is

termed

Magnetic

selection

and

screening

by

FACSMagnetic

selections

were

performed

for

the

first

round

of

selection

using

the

libraries

and

CPX-directed

An

overnight

culture

of

cells

corresponding

to

the

library

diversity

was

inoculated

to

LB

medium

containing

µg/ml

chloramphenicol

-LRB-

Cm

-RRB-

for

a

final

cell

concentration

of

or

µl

of

overnight

cultures

into

ml

LB

Cm

which

ever

was

greater

The

cultures

were

then

grown

at

°C

to

with

shaking

-LRB-

rpm

-RRB-

at

which

time

the

culture

was

moved

to

room

temperature

-LRB-

°C

-RRB-

to

equilibrate

and

then

induced

with

l-arabinose

to

a

final

concentration

of

-LRB-

w/v

-RRB-

The

cells

were

induced

by

shaking

-LRB-

rpm

-RRB-

for

min

at

room

temperature

A

volume

of

cells

corresponding

to

the

library

diversity

was

concentrated

by

centrifugation

-LRB-

g

°C

min

-RRB-

and

resuspended

in

cold

PBS

to

Dynal

MyOne

SA

beads

were

added

to

a

ratio

of

approximately

one

bead

per

four

cells

Magnetic

separation

was

used

to

wash

the

beads

four

times

with

a

volume

of

LB

equivalent

to

the

volume

used

in

the

initial

labeling

and

the

beads

plus

bound

cells

were

finally

resuspended

in

LB

with

Cm

and

glucose

-LRB-

w/v

-RRB-

for

overnight

growth.For

flow

cytometric

sorting

µl

of

overnight

cultures

of

the

libraries

were

inoculated

to

ml

LB

Cm

Cells

were

induced

as

described

in

the

previous

paragraph

in

future

rounds

of

sorting

the

induction

time

was

decreased

to

min

Ten

microliter

of

cells

were

labeled

with

µl

of

nM

SA

PE

in

PBS

on

ice

for

min

pelleted

by

centrifugation

and

the

supernatant

was

removed

Cells

were

resuspended

in

ice-cold

PBS

at

approximately

cells/ml

and

immediately

analyzed

and

sorted

using

a

FACSAria

cytometer

with

nm

excitation

Between

and

of

the

most

labeled

cells

were

collected

and

amplified

for

further

rounds

of

analysis

and/or

sorting

by

growing

overnight

in

LB

medium

containing

glucose

and

Cm

A

subset

of

the

sort

was

plated

directly

on

agar

for

isolation

of

single

clones

Typically

selected

clones

were

assayed

for

antigen

binding

by

flow

cytometry

and

the

identity

of

each

peptide

insert

was

determined

by

DNA

sequencing.Determination

of

peptide

display

levelsTo

compare

the

display

level

of

CPX

and

eCPX

as

a

function

of

induction

time

CPX

and

eCPX

cells

were

subcultured

from

overnight

stocks

into

ml

LB

Cm

and

grown

for

h

with

shaking

-LRB-

rpm

-RRB-

at

°C

The

cells

were

then

moved

to

room

temperature

-LRB-

°C

-RRB-

to

equilibrate

and

induced

with

-LRB-

w/v

-RRB-

l-arabinose

still

shaking

at

rpm

Samples

-LRB-

µl

-RRB-

were

taken

prior

to

induction

and

min

after

induction

then

added

to

µl

of

nM

SA

PE

in

PBS

and

incubated

on

ice

for

min

The

cells

were

then

centrifuged

-LRB-

g

min

-RRB-

supernatant

removed

and

resuspended

in

µl

ice

cold

PBS

Cells

were

immediately

analyzed

with

a

FACSAria

using

nm

excitation

and

fluorescence

data

collected

at

nm.To

compare

the

display

of

various

peptides

using

CPX

and

eCPX

cultures

were

started

using

µl

of

overnight

culture

in

ml

LB

Cm

Cultures

expressing

the

mini-Z

domain

SApep

and

were

grown

until

an

of

and

moved

to

room

temperature

-LRB-

-RRB-

to

equilibrate

and

then

induced

with

l-arabinose

to

a

final

concentration

of

-LRB-

w/v

-RRB-

for

min

or

h

for

the

mini-Z

domain

Cultures

expressing

the

CRPpep

and

peptides

were

induced

at

°C

at

an

of

for

min

Induced

cells

-LRB-

µl

-RRB-

were

added

to

µl

of

PBS

containing

the

respective

antigens

at

the

following

concentrations

nM

YPet-Mona

nM

biotinylated

VEGF

nM

biotinylated

CRP

nM

Alexa

labeled

human

IgG

nM

SA

PE

and

nM

tag

monoclonal

IgG

Samples

were

labeled

on

ice

for

min

Biotinylated

samples

were

spun

down

at

g

for

min

and

the

supernatant

removed

Cells

were

resuspended

in

µl

of

PBS

with

nM

SA

PE

and

put

on

ice

for

min

Before

cytometric

analysis

samples

were

spun

down

at

g

for

min

supernatants

removed

and

µl

of

PBS

added

to

resuspend

the

cells

Samples

were

excited

at

nm

fluorescence

data

were

collected

at

nm

for

SA

PE

labeled

samples

and

nm

for

Alexa

labeled

samples

and

YPet

conjugated

samples.For

the

dual

labeling

experiments

cells

were

subcultured

from

overnight

stocks

into

ml

LB

Cm

and

grown

for

h

shaking

-LRB-

rpm

-RRB-

at

°C

The

cells

were

then

induced

with

-LRB-

w/v

-RRB-

l-arabinose

Cells

expressing

were

expressed

for

min

at

°C

and

cells

expressing

were

expressed

for

min

at

°C

Cells

-LRB-

µl

-RRB-

were

labeled

with

µl

of

PBS

with

nM

SA

PE

only

nM

Ypet-Mona

only

or

with

both

probes

simultaneously

The

negative

controls

were

non-displaying

cells

labeled

with

SA

PE

and

Ypet-Mona

The

cells

were

incubated

at

room

temperature

for

min

centrifuged

at

g

for

min

and

supernatant

removed

The

cells

were

left

on

ice

before

resuspension

with

µl

ice

cold

PBS

and

analyzed

using

cytometry

with

nm

excitation

and

measuring

emission

at

and

nm

Supplementary

dataSupplementary

data

are

available

at

PEDS

online

FundingNational

Science

Foundation

CAREER

award

-LRB-

-RRB-

to

P.S.D.

U.S.

Army

Research

Office

Institute

for

Collaborative

Biotechnologies

-LRB-

-RRB-

Supplementary

Material

-LSB-

Supplementary

Data

-RSB-

B I O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

bacterial display
circular permutation
directed evolution
ompx
peptide display


Endocytic pathways : combined scanning ion conductance and surface confocal microscopy study We introduce a novel high resolution scanning surface confocal microscopy technique that enables imaging of endocytic pits in apical membranes of live cells for the first time . The improved topographical resolution of the microscope together with simultaneous fluorescence confocal detection produces pairs of images of cell surfaces sufficient to identify single endocytic pits . Whilst the precise position and size of the pit is detected by the ion conductance microscope , the molecular nature of the pit , e.g. clathrin coated or caveolae , is determined by the corresponding green fluorescent protein fluorescence . Also , for the first time , we showed that flotillin 1 and 2 can be found co-localising with ~ 200-nm indentations in the cell membrane that supports involvement of this protein in endocytosis . Introduction The role of biological cell plasma membrane endocytic pits , e.g. clathrin-coated pits -LSB- 19 -RSB- and caveolae -LSB- 15 -RSB- , has been extensively studied over the last decades and still remains a hot topic . They not only provide cells with a means of transport across the membrane but also work as signalosomes aggregating receptors and ion channels -LSB- 4 , 17 -RSB- . More recently , another membrane-microdomain-associated protein flotillin -LRB- 1 and 2 -RRB- , traditionally thought to be associated with various receptors such as insulin receptor -LSB- 1 -RSB- , has been shown to be responsible for clathrin - and caveolins-independent endocytosis -LSB- 6 -RSB- , providing an alternative route of trafficking through the cell membrane . Whilst it is well known that clathrin and caveolin form basket-shaped pits in cell membranes -LSB- 17 , 25 -RSB- , the surface structures created by flotillin remain unknown . Current methods for endocytic pit function and dynamics studies include confocal microscopy , total internal reflection -LRB- TIR -RRB- microscopy -LSB- 6 , 20 , 26 -RSB- , scanning and transmission electron microscopy -LSB- 25 -RSB- and atomic force microscopy -LSB- 5 -RSB- . Although fluorescence confocal microscopy is the one of the most commonly used microscopy techniques in cell biology , it provides limited information about single endocytic pit due to its lack of resolution . TIR has better vertical resolution and signal-to-noise ratio and has been used for single endocytic event following -LSB- 2 -RSB- . However , TIR is limited to observation of events that occur on the basal cell membrane only since it relies on imaging to a depth of about 100 nm from the glass surface and is not applicable to studying apical cell surfaces . In addition , using fluorescence-based microscopy techniques , it is impossible to determine the precise location of the endocytic pit relative to the cell membrane since the membrane position is hard to define . On the other hand , electron microscopy techniques have more than sufficient resolution and provide much more detailed information about the surface topography than optical images since the cell membrane can be imaged . However , these require fixation and specialised treatments of the cells , which potentially can alter the structure of interest and are thus incompatible with live studies . Scanning probe microscopy techniques may address a number of these issues by providing topographical data of surfaces . Atomic force microscopy has been used to study cell surfaces but , due to the direct interaction of its probe with the sample , imaging soft and mobile cell surfaces is difficult , limiting most studies to fixed cells or isolated membrane leaflets which are more rigid -LSB- 5 -RSB- . Scanning ion conductance microscopy -LRB- SICM -RRB- , which uses a glass micropipette as an imaging probe -LSB- 9 -RSB- , has no such disadvantage and allows non-contact visualisation of the topography of living cells -LSB- 13 , 14 -RSB- . This has recently been combined with simultaneous confocal fluorescence imaging , resulting in a new technique -- scanning surface confocal microscopy -LRB- SSCM -RRB- -LSB- 7 -RSB- . Also , since its inception , the resolution of the technique has been significantly improved , making it possible to visualise structures as small as protein complexes on surfaces of live cells -LSB- 23 -RSB- . This novel technique allows us to identify fluorescence located on the surface of the cell and localise it with topographically observed nanometre-scale structures -LRB- unpublished results -RRB- . Materials and methods SSCM SSCM is based on a combination of SCM and SICM . SICM is a scanning probe microscopy technique -LSB- 14 , 23 -RSB- in which the ion current flowing into a nanopipette is used to control the vertical -LRB- z axis -RRB- position of the cell relative to the pipette tip . As shown diagrammatically in Fig. 1a -LRB- not to scale -RRB- , in SSCM , the cell is moved up and down in the z direction while scanning in the x and y directions , so its surface is always the same distance from the nanopipette . A laser is passed up a high numerical aperture objective so that it is focused just at the tip of the nanopipette , and a pinhole is positioned at the image plane so that the confocal volume is just below the pipette , as described -LSB- 22 -RSB- . Thus , a fluorescence image of the cell surface is obtained in a single scan , as well as a simultaneously captured image of the cell topography . Fig. 1Topographical imaging of endocytic pits in living cells by SICM . a Schematic diagram of the scanning ion conductance microscope . b SICM topographical image of live Cos-7 cell . c High resolution topographical SICM image of live Cos-7 cell membrane revealing numerous pits . d High resolution topographical SICM image of a fixed Cos-7 cell membrane revealing numerous pits . e Zoomed image showing a single pit -LRB- top -RRB- . Topographical profile of a pit in a live cell -LRB- bottom -RRB- . f Zoomed image showing two pits -LRB- top -RRB- . Topographical profile of pits in fixed cell -LRB- bottom -RRB- The SCIM scanning head was developed in collaboration with Ionscope Limited , UK and mounted on a Nikon TE2000-U Inverted Microscope -LRB- Nikon Co. , Japan -RRB- . The sample holder was attached to a 100-μm HERA XY Nanopositioning System -LRB- Physik Instrumente -LRB- PI -RRB- GmbH & Co. , Germany -RRB- used for lateral scanning . Vertical measurement and modulation was provided by 12-μm LISA XY Nanopositioning System -LRB- Physik Instrumente -LRB- PI -RRB- GmbH & Co. , Germany -RRB- . Both piezo stages were mounted on 25-mm translation stage DC motors -LRB- Physik Instrumente -LRB- PI -RRB- GmbH & Co. , Germany -RRB- to provide coarse lateral and vertical approach . The setups were controlled via a computer with a SBC6711 DSP board equipped with A4D4 ADC/DAC modules -LRB- Innovative Integration , USA -RRB- using SICM software v. 1.2.00 -LRB- Ionscope Limited -RRB- . The time to acquire a 512 × 512 pixel image was approximately 10 min . Two types of nanopipettes were used for the experiments . For low resolution images , nanopipettes with internal diameters ~ 150 nm were pulled from borosilicate glass capillaries . High resolution imaging was made using quartz nanopipettes with internal diameters ~ 70 nm . The nanopipettes were made from 1.00-mm outer diameter by 0.5-mm inner diameter capillaries with inner filament -LRB- Sutter Instrument , USA -RRB- using a laser-based Brown -- Flaming puller -LRB- model P-2000 , Sutter Instrument , San Rafael , CA , USA -RRB- . The nanopipettes , backfilled with phosphate-buffered saline -LRB- PBS -RRB- and lowered in PBS , produced a resistance of approximately 300 MΩ for quartz and 100 MΩ for borosilicate pipettes . The maximum ion current measured using an Axopatch 200B -LRB- Axon Instruments , USA -RRB- was ~ 0.7 nA for quartz and ~ 1.5 nA for borosilicate pipettes . The set-point for imaging was 1 % of the maximum of modulated ion current . The excitation light source was provided by a GPNT-02 laser diode -LRB- 532-nm wavelength , IQ1A 635-nm laser ; Power Technology Inc. , USA -RRB- . The optical recording system consisted of a Nikon TE2000-U Inverted Microscope equipped with a × 100 1.3-N.A. oil-immersion objective . The excitation light was fed through an epi-fluorescent filter block and emitted light was collected by a photomultiplier with a pinhole -LRB- model D-104-814 ; Photon Technology International , UK -RRB- . Image processing and data analysis Matching VLP topographical structure to its corresponding fluorescent signal was done as follows : fluorescent confocal images were threshold to subtract the background , and the positions of individual fluorescent spots were marked by arrowheads and the multiple spots -LRB- where individual signals could not be resolved -RRB- circled . All positional markers were then grouped into one template and placed over the simultaneously recorded topographical image . As the result of this procedure , those topographical features having corresponding fluorescent signals were marked . Image contrast enhancement included slope correction and high-pass filtration performed similarly as previously described -LSB- 10 , 18 -RSB- . The slope was calculated and subtracted from images by least squares algorithm -LSB- 21 -RSB- resulting in image flattening . A high-pass filter is a filter that passes high frequencies efficiently but reduces frequencies lower than the certain specified -LRB- cutoff -RRB- frequency . When applied to a 3D image , high-pass filtering results in finer , i.e. high frequency , details to stand out because larger features are eliminated . Cell culture and plasmids Monkey Cos-7 cells were routinely maintained at 37 °C in 5 % CO2 , using DMEM -LRB- GIBCO/BRL -RRB- containing 5 % -LRB- vol . / vol . -RRB- FCS . The plasmid DNA used in the experiments were in pCi -LRB- Promega -RRB- containing either caveolin-green fluorescent protein -LRB- GFP -RRB- , clathrin-GFP , flotillin-1-GFP or flotillin-2-GFP . Transfection procedure Cos-7 cells -LRB- 1 × 106 cells per flask -RRB- were plated into a T25 flask and incubated overnight at 37 °C in DMEM containing 5 % FCS . Cells were washed prior to transfection with PBS , and complexes of Lipofectamine -LRB- Invitrogen -RRB- and plasmid DNA at a ratio of 1 μl to 1 μg were added in Optimem -LRB- Invitrogen -RRB- without FCS to the cells . For flotillin transfections , a mixture of flotillin-1 - and flotillin-2-GFP plasmids were complexed together and added to the cells . After 2 h , the media was replaced with DMEM containing 5 % FCS . After 24 h , the cells were trypsinised and plated onto coverslips -LRB- 5 × 104 per well -RRB- and incubated at 37 °C overnight . Cells where then either used for live imaging or fixed for 20 min with 3 % formaldehyde containing 5 % sucrose . Results Imaging of the cell surface using SICM In order to access the apical surface of Cos-7 cells , we used a basic arrangement of the SICM . A schematic diagram of the microscope is presented in Fig. 1a -LRB- not to scale -RRB- , while a detailed description of the instrument can be found in `` Materials and methods '' section . SICM -LSB- 9 , 13 , 14 -RSB- is a type of scanning probe microscopy that uses a glass nanopipette as a sensitive probe to acquire a topographical image of the surface . Distance feedback-control is based on the ion current that flows between electrodes in the nanopipette and the bath , and this current is reduced as the pipette approaches the surface . This allows non-contact imaging of live cells in a physiological buffer . Using the SICM technique , we are able to either examine the morphology of the whole cell or achieve high resolution images of the cell surface . Scanning the complete cell surface gives insight into the number of macrostructures present on the cell surface , for example microvilli , and can be used to probe cell surface dynamics -LSB- 8 -RSB- . The morphology of the cell also has an impact on the resolution of SICM images , since large structures and steep slopes make small features difficult to resolve . A typical SICM image of live untransfected -LRB- control -RRB- Cos-7 cell in physiological conditions is presented in Fig. 1b . Cos-7 cells have very few macrostructures visible on the surface , and although the highest part of the cell membrane above the nucleus -LRB- light area , Fig. 1b -RRB- generates a steep slope , there are also flat peripheral areas . High resolution scans of these peripheral areas generate images containing a number of indentations of the cell surface -LRB- Fig. 1c -RRB- . Observed indentations range from 50 - to 160-nm diameters -LRB- see an example in Fig. 1e -RRB- and could present endocytic pits formed by clathrin and/or caveolin . The mobility of these indentations was relatively high compared to the present scan rate of our SICM as shown later in the paper ; therefore , we used fixed cells to identify particular endocytic pits and imaged them at a higher resolution . A high resolution image of a fixed Cos-7 cell presented in Fig. 1d reveals similar indentations as seen in Fig. 1c but at higher clarity and contrast . A digitally zoomed image is presented in Fig. 1f and shows two pits -LRB- top -RRB- with the corresponding topographical profiles -LRB- bottom -RRB- . Fig. 2Topographical and fluorescent imaging of clathrin coated pits in fixed clathrin-GFP transfected Cos-7 cells by scanning surface confocal microscope . a Schematic diagram of SSCM . b Topographical image of cell . c Fluorescent image of the clathrin-GFP transfected cell shown in b. d 3D representation of overlaid topographical and fluorescent images shown in b and c , respectively . e High resolution topographical image of cell surface revealing numerous clathrin-coated pits . f Same topographical image as in e but inverted and presented in a red palette . g Overlaid inverted topographical and fluorescent images shown in f and g , respectively . The image reveals that the pit topography matches the clathrin-GFP fluorescence . h The distribution of clathrin-coated pit width calculated from the SICM topographical images Scanning surface confocal microscopy In order to identify what pit indentations detected topographically by SICM corresponded to what endocytic pits , clathrin or caveolin , we transfected Cos-7 cells with corresponding GFP construct and then used scanning surface confocal microscopy to study the transfected cells . A schematic diagram of the microscope is presented in Fig. 2a -LRB- not to scale -RRB- . Basically , SSCM is a combination of SICM and scanning confocal microscope -LSB- 7 -RSB- . SSCM also uses a glass nanopipette to acquire a true topographical image of the surface and a combination of a laser , pinhole and photomultiplier to gather fluorescent information from the cell surface , removing any out-of-focus fluorescence . Because the laser beam is always focused on the very tip of the nanopipette , topographical and fluorescent data are spatially aligned , thus enabling correlation between topographical features and fluorescent signals to be found . Imaging clathrin-coated pits in membranes of fixed cells using SSCM Cells transfected with clathrin-GFP were scanned , at low and high resolution , to examine the distribution of clathrin-coated pits on the cell surface . A typical topographical image of clathrin-FGP transfected fixed Cos-7 cell , together with its fluorescent image , is presented in Fig. 2b and c , respectively . An overlay of topographical and fluorescent images is shown in Fig. 2d . Peripheral areas of the cell contain punctated GFP fluorescence and had a flat topography . A high resolution topographical scan of these cell surfaces at a 7 × 7-μm scan -LRB- Fig. 2e -RRB- reveals numerous indentations ranging from 58 to 159 nm with an average depth of 102 nm -LRB- see distribution in Fig. 2h -RRB- . In order to identify what proportion of the imaged pits were real clathrin-coated pits , we overlaid the topographical and fluorescent data . To make pit indentations that appear as dark spots in topographical images , visible when overlaid with the fluorescence image , the topographical image was inverted and red palette was applied -LRB- Fig. 2f -RRB- . An overlaid , inverted topography red palette and clathrin-GFP fluorescence image is presented in Fig. 2g . As can be seen , there is a ` cloud ' of green fluorescent signal around almost every single red spot that corresponds to indentations in the topography , giving a high degree of co-localisation between these structures and fluorescent puncta . Therefore , we can conclude that the majority of observed indentations are clathrin-coated pits . Analysis of the images revealed that 102 out of 115 -LRB- 88.7 % -RRB- detected endocytic pits co-localised with clathrin-GFP fluorescent spots . Presumably , this is because we are also detecting other types of pits that are not clathrin-coated . Reverse analysis showed that 11 out of 113 -LRB- 9.7 % -RRB- detected clathrin-GFP fluorescence spots did not match pit indentations . This could reflect the fact that clathrin-coated pits become highly mobile and move away from the surface inside the cell as soon as they bud off the cell membrane -LSB- 16 -RSB- . At higher magnification , we could also see clustering of the clathrin-coated pits in round patches -LRB- data not shown -RRB- that is similar to observations made by Frankel et al. -LSB- 5 -RSB- . Imaging caveolae in membranes of fixed cells using SSCM Using SSCM , the surface of caveolin-GFP transiently transfected cells was then examined . Low resolution topographical , fluorescent and overlaid images are presented in Fig. 3a , b and c , respectively . Higher resolution studies -LRB- Fig. 3d and e -RRB- revealed indentations in the cell membrane . These pits were slightly smaller than the clathrin-coated pits , ranging between 52 and 90 nm and giving a mean depth of 69 nm -LRB- see distribution in Fig. 3f -RRB- . Fig. 3Topographical and fluorescent imaging of caveolin pits in fixed caveolin-GFP transfected Cos-7 cells by SSCM . a Topographical image of cell . b. Fluorescent image of caveolin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b , respectively . d High resolution topographical image of cell surface . e High resolution fluorescent image of caveolin-GFP transfected cell shown in d. f Caveolin pit width distribution histogram calculated from SICM topographical images . g Digital zoom of the topographical image shown in d -LRB- dotted square -RRB- revealing numerous caveolin pits . h Same zoomed topographical image as in g but inverted and presented in a red palette . i Overlaid inverted topographical image shown in h and digitally zoomed fluorescent image from the area shown in e -LRB- dotted square -RRB- . The image reveals that pits match the caveolin-GFP fluorescence On closer examination using digitally zoomed images -LSB- topographical -LRB- Fig. 3g -RRB- , topographical inverted and in red palette -LRB- similar to one described for clathrin -RRB- -LRB- Fig. 3h -RRB- and an overlay of the topography and fluorescence image -LRB- Fig. 3i -RRB- -RSB- , it could be seen that the indentations in the cell surface co-localise with fluorescent signal . However , compared to clathrin-coated pits , identification of caveolae is more problematic . In all experiments conducted , the fluorescent images have a punctate appearance . However , in only two out of 26 experiments did fluorescence and structure co-localise . In those experiments , 139 pits were observed on the cell surface and only 13 of them -LRB- 9.35 % -RRB- were identified as matching caveolin-GFP fluorescent signal . This is in good agreement with the previous measurement that 90 % of the pits identified in the topographic image on the cell surface are clathrin-coated and indicates that the other 10 % are caveolae . Our observation that we detect many fluorescence spots from caveolin-GFP that do not correspond to pits in the SICM image suggests that many caveolae are below the cell surface or present on the surface but not resolvable . By transmission electron microscopy , they have been identified as 50 -- 100 nm in size -LSB- 15 -RSB- ; however , this is often the size of the whole structure , with the neck being much smaller -LSB- 25 -RSB- . It has even been suggested that there is a proteinaceous cap covering the neck of the caveolae -LSB- 25 -RSB- . If this is the case , and the cap could influence the flow of ions , then we would not be able to image caveolae structures on the cell surface unless the cap is open . Imaging flotillin in membranes of fixed cells using SSCM To our knowledge , flotillin 1 and 2 has not been yet associated with cell membrane structure that could be detected topographically by any microscopy technique . However , it has been shown that this protein is present in lipid rafts -LSB- 1 -RSB- and is responsible for clathrin - and caveolin-independent endocytosis -LSB- 6 -RSB- . Here , we examined Cos-7 cells transfected with flotillin 1 and 2 by SSCM for the presence of topographically detectable membrane features that could potentially co-localise with flotillin-GFP fluorescent signal . Low resolution topographical and confocal images together with overlaid image of flotillin-GFP-transfected Cos-7 cells are presented in Fig. 4a , b and c , respectively . Higher-resolution topographical images of the cell surface -LRB- Fig. 4d and g -RRB- revealed 200 ± 20-nm indentations . Figure 4e presents inverted red palette topographical image of Fig. 4d . Inverted red palette topography and fluorescence overly reveals that some indentations co-localise with the flotillin-GFP fluorescent signal -LRB- Fig. 4f , arrows -RRB- . Remarkably , we have found that larger and higher-intensity fluorescent spots correlated with smooth protrusions from the cell surface -LRB- Fig. 4g , h and i , hollow arrows -RRB- . The image presented in Fig. 4h is a high-pass filtered topographical image of the same data as in Fig. 4g . Fig. 4Topographical and fluorescent imaging of fixed flotillin-GFP transfected Cos-7 cells by SSCM . a Topographical image of cell . b. Fluorescent image of flotillin-GFP transfected cell shown in a. c 3D representation of overlaid topographical and fluorescent images shown in a and b , respectively . d High resolution topographical image of the cell surface revealing numerous indentations . e Same topographical image as in d but inverted and presented in red palette . f Overlaid inverted topographical image shown in e and high resolution fluorescent image of flotillin-GFP acquired from the same area . The image reveals that some indentations on the cell surface match flotillin-GFP fluorescence -LRB- white arrows -RRB- . g High resolution topographical image of cell surface revealing numerous indentations -LRB- solid arrows -RRB- as well as two protrusions -LRB- hollow arrows -RRB- . h Same topographical image as in g but high-pass filtered . i High resolution fluorescent image of caveolin-GFP transfected cell shown in g . The arrows point to indentations that match flotillin-GFP fluorescence . Hollow arrows point to protrusions that match flotillin-GFP fluorescence Imaging endocytic pits in membranes of living cells using SSCM In order to test whether SSCM can identify particular endocytic pits in membranes of live cells , we performed a series of experiments with live clathrin-GFP transfected Cos-7 cells . Figure 5a and b presents normal and inverted red palette topographical images of a live cell . When overlaid with fluorescence , the inverted red palette topography shows that almost all topographically detected pits co-localise with clathrin-GFP fluorescence . There are fluorescence spots that are not round , but elongated in shape that do not match pit indentations on the surface . These spots of fluorescence probably reflect fast-moving clathrin vesicles right under the cell membrane . Fig. 5Live topographical and fluorescent imaging of clathrin coated pits in clathrin-GFP transfected Cos-7 cells by SSCM . a High resolution topographical image of live cell membrane revealing numerous clathrin-coated pits . b. Same topographical image as in a but inverted and presented in red palette . c Overlaid inverted topographical image shown in a and fluorescent image of the same area . The image reveals that , on live cells , we can detect that the pits ' topography match clathrin-GFP fluorescence . d Sequence of topographical images of live cell membrane revealing dynamics of the clathrin-coated pits . The images are separated by 10 min Figure 5d shows a sequence of three topographical images acquired from the same area of a cell with 10-min intervals . As can be seen , the indentations that correspond to endocytic pits are highly mobile and appear on or disappear from the surface of the cell membrane . It is beyond the current time resolution of our SSCM to follow the dynamics of these pits . However , this is the first time that endocytic pits are resolved topographically on the surface of live cell . Discussion By combining high resolution ion conductance imaging of the cell surface topography with fluorescence confocal imaging , we can identify the molecular nature of endocytic pits on the surface of living cells and measure the topography of the pits . For the first time , we showed that flotillin 1 and 2 is involved in the formation of ~ 200-nm-size indentations in the cell membrane . This observation is important evidence in support of the involvement of this protein in clathrin - and caveolin-independent endocytosis . We have found on Cos-7 cells that about 89 % of the detected pits are clathrin-coated and 9 % are caveolae , leaving a small percentage to be presented by flotillin pits . In each particular case , cell preparation transfection could introduce some deviation of clathrin/caveolin/flotillin proportion comparing to untransfected control . The fact that clathrin-coated pit formation is dependent on multiple factors -LSB- 11 , 24 -RSB- provides indirect evidence that transfection may not influence the amount of pit formation . In contrast , it has been shown that expressing the caveolin in cells that do not contain this protein is enough to form caveolae -LSB- 3 -RSB- . However , there are other studies indicating that , although in caveolin transfected cells the total amount of produced caveolin is increased , the concentration of caveolin in the cell membrane remains unchanged -LSB- 12 -RSB- . The sizes of the pits we have measured are in good agreement with those obtained by electron microscopy . We have also shown that it is possible to apply our method to live cells . The clathrin-coated pits show fast dynamics on the time scale of our current imaging but suggest that improvements in the speed of imaging should allow us to follow the dynamics of endocytic pits on living cells with the intriguing possibility of directly imaging the endocytic process in real time .

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O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

caveola
membrane topology
membrane transport