The

mammalian

centrosome

and

its

functional

significance

Primarily

known

for

its

role

as

major

microtubule

organizing

center

the

centrosome

is

increasingly

being

recognized

for

its

functional

significance

in

key

cell

cycle

regulating

events

We

are

now

at

the

beginning

of

understanding

the

centrosome

's

functional

complexities

and

its

major

impact

on

directing

complex

interactions

and

signal

transduction

cascades

important

for

cell

cycle

regulation

The

centrosome

orchestrates

entry

into

mitosis

anaphase

onset

cytokinesis

transition

and

monitors

DNA

damage

Recently

the

centrosome

has

also

been

recognized

as

major

docking

station

where

regulatory

complexes

accumulate

including

kinases

and

phosphatases

as

well

as

numerous

other

cell

cycle

regulators

that

utilize

the

centrosome

as

platform

to

coordinate

multiple

cell

cycle-specific

functions

Vesicles

that

are

translocated

along

microtubules

to

and

away

from

centrosomes

may

also

carry

enzymes

or

substrates

that

use

centrosomes

as

main

docking

station

The

centrosome

's

role

in

various

diseases

has

been

recognized

and

a

wealth

of

data

has

been

accumulated

linking

dysfunctional

centrosomes

to

cancer

Alstrom

syndrome

various

neurological

disorders

and

others

Centrosome

abnormalities

and

dysfunctions

have

been

associated

with

several

types

of

infertility

The

present

review

highlights

the

centrosome

's

significant

roles

in

cell

cycle

events

in

somatic

and

reproductive

cells

and

discusses

centrosome

abnormalities

and

implications

in

disease

Introduction

Over

years

have

passed

since

the

discovery

of

centrosomes

-LRB-

Flemming

Van

Beneden

-RRB-

and

yet

we

are

only

at

the

beginning

of

understanding

the

multiple

and

central

roles

of

this

intriguing

cellular

organelle

Boveri

-LRB-

-RRB-

who

coined

the

name

centrosomes

had

produced

a

remarkable

wealth

of

new

information

characterizing

and

analyzing

centrosomes

during

fertilization

and

cell

division

-LRB-

Boveri

-RRB-

by

using

iron

hematoxylin

as

a

major

cytological

staining

technique

and

he

brilliantly

recognized

the

very

important

role

of

centrosomes

in

cancer

-LRB-

-RRB-

Flemming

had

called

the

discovery

of

centrosomes

as

important

as

the

discovery

of

the

nucleus

-LRB-

Flemming

-RRB-

yet

research

on

centrosomes

stagnated

for

decades

for

a

number

of

reasons

including

war

events

in

Europe

that

slowed

all

research

and

because

studies

using

the

electron

microscope

did

not

reveal

a

structure

that

could

be

easily

defined

Centrosome

structure

was

described

as

amorphous

osmiophilic

material

surrounding

a

pair

of

well-structured

centrioles

which

stimulated

and

generated

new

research

on

centrioles

while

research

on

centrosomes

became

less

appreciated

With

the

development

of

immunological

probes

and

molecular

techniques

coupled

with

immunofluorescence

microscopy

centrosomes

reclaimed

their

status

and

Boveri

's

earlier

work

was

vigorously

pursuit

by

reproductive

biologists

and

by

cellular

and

cancer

biologists

who

were

able

to

confirm

and

build

on

Boveri

's

remarkably

detailed

and

accurate

data

and

visionary

research

Our

understanding

of

centrosomes

has

advanced

enormously

and

very

rapidly

in

recent

years

and

its

central

role

in

cellular

biology

has

clearly

been

recognized

It

can

be

predicted

that

a

number

of

diseases

other

than

cancer

are

linked

directly

or

indirectly

to

centrosome

dysfunctions

Centrosome

isolations

forward

and

reverse

genetics

RNA-mediated

interference

mass-spectrometry-based

proteomics

live

cell

imaging

particularly

with

GFP-tagged

proteins

and

laser

microsurgery

are

some

of

the

newer

more

recently

employed

techniques

that

have

been

applied

to

centrosome

research

and

rapidly

moved

the

field

into

new

frontiers

The

present

paper

will

review

the

state

of

our

knowledge

of

centrosomes

and

analyze

its

impact

on

cellular

functions

Definition

of

the

centrosome

and

its

key

functions

The

centrosome

is

a

subcellular

non-membrane

bound

semi-conservative

organelle

approximately

μm

in

size

that

serves

as

the

cell

's

primary

microtubule

organizing

center

-LRB-

MTOC

-RRB-

and

plays

a

major

role

in

numerous

cellular

functions

Through

its

microtubule

organizing

functions

the

centrosome

facilitates

many

cellular

activities

including

cell

motility

polarity

maintenance

of

cell

shape

cell

division

transport

of

vesicles

and

targeting

of

numerous

signaling

molecules

A

typical

mammalian

centrosome

consists

of

a

proteinaceous

scaffold

containing

a

large

number

of

centrosome

proteins

including

γ-tubulin

and

the

gamma-tubulin

complex

-LRB-

γ-TuRC

-RRB-

that

typically

surround

a

pair

of

perpendicularly

oriented

cylindrical

centrioles

therefore

referred

to

as

pericentriolar

material

-LRB-

PCM

-RRB-

The

PCM

scaffold

undergoes

shape

changes

throughout

the

cell

cycle

to

produce

cell-cycle-specific

microtubule

organizations

for

specific

cellular

functions

Lacking

a

defined

membrane

boundary

the

centrosome

's

three-dimensional

architecture

is

maintained

through

specific

protein

protein

interactions

Microtubules

are

anchored

at

the

centrosome

with

their

minus

ends

-LRB-

Bornens

-RRB-

and

microtubule

growth

is

regulated

by

distal

plus-end

addition

of

tubulin

subunits

-LRB-

McIntosh

and

Euteneuer

-RRB-

In

interphase

the

centrosome

is

juxtapositioned

and

closely

associated

with

the

nucleus

At

the

transition

it

undergoes

significant

cell

cycle-specific

reorganizations

and

matures

into

the

division-competent

center

of

the

mitotic

poles

Because

the

centrosome

nucleates

microtubules

and

controls

microtubule

numbers

and

lengths

it

directs

most

microtubule-related

functions

including

transport

of

macromolecular

complexes

positioning

of

cell

organelles

cell

motility

cell

shape

polarity

and

segregation

of

chromosomes

during

cell

division

The

mitotic

centrosomes

are

critically

important

to

mediate

the

strictly

balanced

bipolar

separation

of

chromosomes

In

addition

to

the

regular

function

as

MTOC

the

centrosome

orchestrates

various

major

important

cell

cycle

events

including

entry

into

mitosis

anaphase

onset

cytokinesis

transition

and

monitoring

of

DNA

damage

-LRB-

Kramer

et

al.

-RRB-

While

a

typical

centrosome

consists

of

a

pair

of

centrioles

surrounded

by

PCM

centrioles

are

not

always

present

within

the

centrosome

structure

as

will

be

detailed

later

in

this

paper

In

general

centrioles

are

important

for

centrosome

integrity

and

to

aid

in

the

recruitment

of

centrosomal

proteins

into

the

PCM

The

PCM

is

a

fibrous

scaffolding

lattice

that

consists

of

a

large

amount

of

coiled-coil

centrosome

proteins

and

anchors

signaling

molecules

and

components

of

the

γ-TuRC

-LRB-

Fig.

-RRB-

Some

centrosome

proteins

are

permanently

associated

with

the

centrosome

core

structure

such

as

γ-tubulin

the

γ-TuRC

and

centrin

-LRB-

addressed

below

in

more

detail

-RRB-

while

several

other

centrosomal

proteins

are

temporarily

associated

with

the

centrosome

core

structure

and

include

centrosome

proteins

such

as

the

Nuclear

Mitotic

Apparatus

protein

-LRB-

NuMA

-RRB-

Such

centrosomal

proteins

serve

cell-cycle-specific

functions

and

are

needed

for

specific

cell

cycle

activities

The

cell

cycle-specific

centrosome

proteins

are

different

from

those

proteins

or

enzymes

-LRB-

kinases

phosphatases

and

others

-RRB-

that

may

be

colocalized

with

centrosomes

and

may

utilize

centrosomes

as

a

docking

station

but

are

not

centrosome

proteins

that

are

directly

involved

in

performing

centrosome-specific

functions

So

far

about

different

proteins

have

been

associated

with

the

centrosome

by

mass

spectroscopy

-LRB-

Andersen

et

al.

-RRB-

although

it

is

not

clear

whether

all

of

those

proteins

are

centrosome

proteins

or

proteins

or

enzymes

that

use

centrosomes

as

docking

station

Regulators

of

various

cell

cycle

events

have

been

co-localized

with

centrosomes

and

include

the

mitogen

activated

protein

kinase

-LRB-

MAPK

-RRB-

-LRB-

Sun

et

al.

-RRB-

which

may

use

centrosomes

as

a

docking

station

to

perform

and

coordinate

multiple

cell

cycle-specific

functions

Vesicles

that

are

translocated

along

microtubules

to

and

away

from

centrosomes

may

also

carry

enzymes

or

substrates

that

use

centrosomes

as

docking

platform

-LRB-

Fig.

b

-RRB-

Fig.

typical

mammalian

centrosome

is

composed

of

two

centrioles

surrounded

by

a

meshwork

of

a

proteins

embedded

in

matrix

called

the

pericentriolar

material

-LRB-

PCM

-RRB-

Gamma-tubulin

and

the

gamma-tubulin

ring

complex

that

nucleate

microtubules

along

with

associated

proteins

are

embedded

in

the

PCM

Highlighted

in

this

diagram

are

two

centrosomal

complexes

the

microtubule

nucleating

complex

and

the

microtubule

anchoring

complexFig

The

centrosome

serves

as

central

station

to

mediate

substrate

and

enzyme

activities

along

microtubules

towards

the

minus

ends

of

microtubules

driven

by

dynein

-LRB-

white

circles

-RRB-

and

toward

the

plus

ends

driven

by

kinesin

-LRB-

black

circles

-RRB-

b

Through

its

microtubule-organizing

capabilities

the

centrosome

mediates

distribution

of

cell

organelles

such

as

mitochondria

Golgi

and

vesicles

of

various

sizes

many

containing

regulatory

enzymes

for

cell

cycle

regulation

Functional

homologues

of

centrosomes

are

spindle

pole

bodies

of

yeast

-LRB-

Knop

et

al.

-RRB-

the

nucleus-associated

body

of

the

cellular

slime

mold

-LRB-

Daunderer

et

al.

-RRB-

and

nucleating

sites

in

plants

-LRB-

Chan

et

al.

-RRB-

In

addition

multiple

plasma

membrane-associated

sites

in

Drosophila

epidermal

cells

-LRB-

Mogensen

-RRB-

and

in

Xenopus

oocytes

-LRB-

Pfeiffer

and

Gard

-RRB-

have

also

been

identified

to

serve

as

MTOCs

New

research

has

established

that

virus

bacteria

and

parasites

exploit

the

host

cell

's

centrosomal

capabilities

and

recruit

centrosomal

material

for

their

own

survival

within

host

cells

-LRB-

reviewed

in

Scaplehorn

and

Way

Coppens

et

al.

-RRB-

Taken

together

the

centrosome

is

a

truly

central

and

main

cellular

station

that

directs

coordinates

and

regulates

most

cellular

functions

either

directly

or

indirectly

through

its

microtubule

organizing

capabilities

Centrosome

proteins

and

their

specific

functions

Numerous

centrosome

proteins

have

been

described

and

their

functions

have

been

determined

but

the

list

of

newly

discovered

centrosome

proteins

continues

to

grow

and

includes

centrosome

proteins

that

have

multifunctional

capabilities

Furthermore

the

list

of

diseases

in

which

centrosome

protein

dysfunctions

play

a

role

also

continues

to

grow

as

research

on

centrosomes

has

progressed

rapidly

and

new

research

teams

have

formed

around

the

growing

centrosome

field

The

initial

discovery

of

centrosomal

proteins

in

an

autoimmune

antibody

has

provided

a

most

powerful

tool

to

stain

centrosomes

with

immunological

probes

-LRB-

Calarco-Gillam

et

al.

-RRB-

and

since

then

a

large

number

of

centrosomal

proteins

have

been

identified

based

on

purification

of

autoimmune

sera

and

subsequent

production

of

specific

centrosome

antibodies

The

reported

number

of

centrosome

proteins

varies

depending

on

the

methods

applied

Because

the

centrosome

is

a

non-membrane-bound

organelle

it

does

not

have

defined

borders

and

isolation

methods

can

be

flawed

by

removing

critical

centrosome

proteins

or

including

contaminants

that

are

not

centrosome

proteins

In

addition

because

the

centrosome

is

a

flexible

structure

that

undergoes

cell

cycle-specific

changes

the

number

of

centrosome

proteins

varies

in

different

cell

cycle

stages

Functional

screens

have

been

used

but

because

of

the

nature

of

centrosomes

may

not

detect

all

centrosome

proteins

Mass

spectroscopy

has

identified

as

many

as

centrosomal

proteins

-LRB-

Andersen

et

al.

-RRB-

while

a

more

conservative

number

of

have

been

identified

with

different

methods

at

various

cell

cycle

stages

with

about

being

present

in

the

interphase

centrosome

-LRB-

reviewed

by

Wilkinson

et

al.

-RRB-

and

may

reflect

the

average

centrosome

protein

quantities

that

are

characteristic

for

interphase

centrosomes

in

typical

mammalian

somatic

cells

Various

definitions

have

been

used

to

describe

the

centrosome

It

is

generally

agreed

that

the

centrosome

core

structure

consists

of

permanently

associated

centrosome

proteins

that

remain

after

treatment

of

the

centrosome

complex

with

microtubule

depolymerizing

agents

such

as

cold

nocodazole

colchicine

derivatives

and

others

The

well-studied

γ-tubulin

is

a

protein

of

the

centrosome

core

structure

Centrosome

core

proteins

are

permanently

associated

with

the

centrosome

structure

throughout

cell

cycle-dependent

structural

changes

in

most

systems

Such

cell

cycle-specific

changes

are

particularly

apparent

in

reproductive

cells

after

fertilization

in

which

rapid

changes

of

centrosomes

and

microtubule

formations

are

required

for

rapid

cell

cycle

functions

Other

centrosome

proteins

associate

with

the

core

structure

during

different

cell

cycle

stages

to

perform

cell

cycle-specific

functions

-LRB-

discussed

below

-RRB-

Such

proteins

include

NuMA

-LRB-

reviewed

in

Sun

and

Schatten

-RRB-

that

performs

essential

functions

in

mitosis

The

analysis

of

determining

the

number

of

centrosome

proteins

is

further

complicated

by

the

great

number

of

proteins

that

use

centrosomes

as

a

central

docking

station

or

platform

where

regulatory

complexes

accumulate

and

communicate

by

signaling

through

the

microtubule

network

Such

complexes

may

co-localize

with

centrosomes

but

should

not

be

included

in

the

definition

of

the

centrosome

Known

and

newly

identified

centrosome

proteins

that

localize

to

human

centrosomes

are

reviewed

in

Wilkinson

et

al.

-LRB-

-RRB-

Centrosome

proteins

that

are

known

to

be

present

in

purified

centrosomes

and

identified

by

mass

spectrometric

analysis

include

the

structural

proteins

alpha

tubulin

beta

tubulin

gamma-tubulin

gamma-tubulin

complex

components

centrin

and

pericentrin/kendrin

ninein

pericentriolar

material

-LRB-

-RRB-

ch-TOG

protein

centriole-associated

protein

centriolin

centrosomal

associated

protein

-LRB-

CPAP

-RRB-

CLIP-associating

proteins

and

CLASP

cenexin

Nudel

centractin

myomegalin

the

regulatory

molecules

cell

division

protein

-LRB-

-RRB-

cAMP-dependent

protein

kinase

type

II

alpha

regulatory

chain

cAMP-dependent

protein

kinase

alpha

catalytic

subunit

serine/threonine

protein

kinase

serine/threonine-protein

kinase

serine/threonine-protein

kinase

Sak

Casein

kinase

I

delta

and

epsilon

isoforms

protein

phosphatase

protein

phosphatase

alpha

isoform

proteins

epsilon

and

gamma

isoforms

the

motor

and

related

proteins

dynein

heavy

chain

dynein

intermediate

chain

dynein

light

chain

dynactin

Glued

dynactin

dynactin

and

the

heat

shock

proteins

heat

shock

protein

TCP

subunits

and

heat

shock

protein

Centrosomes

can

be

depleted

of

many

PCM

components

by

treatment

with

high

salt

which

also

extracts

γ-tubulin

-LRB-

Moritz

et

al.

-RRB-

leaving

behind

centrosome

scaffolds

A

centrosome

matrix

has

been

described

in

several

studies

and

has

been

visualized

in

the

classic

sea

urchin

invertebrate

model

system

with

low

voltage

field

emission

scanning

electron

microscopy

-LRB-

LVFESEM

-RRB-

on

cold

treated

and

isolated

centrosomes

-LRB-

Thompson-Coffe

et

al.

reviewed

in

Schatten

et

al.

Schatten

and

Chakrabarti

-RRB-

which

revealed

a

particle

structure

of

μm

and

repeating

subunits

Centrosome

proteins

that

have

been

studied

more

fully

in

various

cell

systems

include

γ-tubulin

pericentrin

centrin

and

NuMA

Gamma-tubulin

is

an

essential

centrosome

protein

which

had

been

discovered

through

fungal

genetics

-LRB-

Oakley

and

Oakley

-RRB-

and

since

then

described

in

all

eukaryotic

cell

groups

Gamma

tubulin

is

not

only

found

in

centrosomes

but

it

also

can

serve

as

nucleating

sites

in

animal

cells

that

do

not

display

typical

centrosomes

-LRB-

Tulu

et

al.

-RRB-

Gamma-tubulin

is

localized

at

the

oocyte

cortex

in

amphibians

which

has

been

shown

by

Pfeiffer

and

Gard

-LRB-

-RRB-

In

plants

that

do

not

have

typical

centrosomes

γ-tubulin

is

thought

to

nucleate

unfocused

microtubules

within

the

cell

and

it

is

found

along

the

length

of

microtubules

-LRB-

Canaday

et

al.

-RRB-

Gamma-tubulin

is

a

highly

conserved

protein

in

eukaryotes

It

is

vital

for

centrosome

functions

and

elimination

of

γ-tubulin

is

lethal

to

cells

and

to

the

organism

-LRB-

Oakley

et

al.

Joshi

et

al.

-RRB-

while

elimination

of

components

that

bind

to

γ-tubulin

has

little

effect

on

cell

viability

The

function

of

γ-tubulin

was

clearly

determined

by

purification

of

the

γ-tubulin

ring

complex

-LRB-

γ-TuRC

-RRB-

that

revealed

a

ring

shape

and

a

substructure

that

were

able

to

nucleate

microtubule

polymerization

in

vitro

-LRB-

Zheng

et

al.

-RRB-

The

ca.

γ-TuRC

consists

of

or

γ-tubulin

molecules

and

is

the

nucleating

complex

for

microtubules

in

all

cells

studied

so

far

-LRB-

Hannak

et

al.

-RRB-

Gamma

tubulins

may

laterally

interact

with

each

other

Several

accessory

proteins

are

associated

with

this

complex

-LRB-

Tassin

and

Bornens

Gunawardane

et

al.

Murphy

et

al.

-RRB-

providing

new

challenges

for

further

investigations

The

γ-TuRC

has

been

well

characterized

by

electron

microscopy

including

immunoelectron

tomography

showing

an

open

ring

structure

of

ca.

nm

diameter

-LRB-

Moritz

et

al.

Zheng

et

al.

Oegema

et

al.

Moritz

et

al.

-RRB-

The

γ-TuRC

is

capable

of

functionally

capping

the

minus

ends

of

microtubules

facilitating

growth

of

microtubule

protofilaments

Six

components

of

the

γ-TuRC

in

addition

to

γ-tubulin

have

been

identified

so

far

-LRB-

Gunawardane

et

al.

Murphy

et

al.

Tassin

et

al.

-RRB-

Gamma-tubulin

complexes

are

required

for

microtubule

assembly

which

has

been

shown

by

using

salt-extracted

centrosomes

that

lack

γ-TuRCs

and

are

not

able

to

recruit

microtubules

while

addition

of

γ-tubulin

complexes

from

cytoplasmic

extracts

restored

nucleation

competency

although

various

other

components

are

needed

to

anchor

the

γ-TuRC

to

the

centrosome

structure

including

the

large

coiled-coil

A-kinase

anchoring

proteins

-LRB-

Tassin

et

al.

Murphy

et

al.

Dictenberg

et

al.

Doxsey

et

al.

Flory

and

Davis

Gillingham

and

Munro

Kawaguchi

and

Zheng

Keryer

et

al.

Steadman

et

al.

Takahashi

et

al.

-RRB-

and

-LRB-

Ohta

et

al.

-RRB-

The

microtubule-minus-end-binding

proteins

including

γ-TuRC

are

concentrated

at

the

proximal

ends

of

centrioles

while

tubulin

polyglutamylation

of

the

centriole

walls

modulates

interaction

between

tubulin

and

microtubule

associated

proteins

The

centriole-interacting

proteins

can

bind

and

aid

in

the

recruitment

of

matrix

proteins

A

cell

cycle-dependent

regulation

of

γ-TuRC

is

thought

to

account

for

differences

between

interphase

and

mitotic

microtubule

structure

formations

and

activities

In

interphase

γ-TuRC

nucleates

fewer

but

longer

microtubules

while

in

mitosis

more

γ-TuRC

is

associated

with

the

centrosome

which

is

part

of

the

centrosome

maturation

process

that

takes

place

from

interphase

to

mitosis

-LRB-

discussed

below

-RRB-

Mitotic

microtubules

are

shorter

larger

in

number

and

highly

dynamic

They

are

regulated

by

a

number

of

cell

cycle-specific

proteins

that

participate

in

centrosome

regulation

such

as

the

small

GTPase

Ran

Aurora

A

kinase

polo-like

kinases

and

others

Pericentrin

is

a

centrosome

protein

that

plays

a

role

in

centrosome

and

spindle

organization

-LRB-

Doxsey

et

al.

Dictenberg

et

al.

Young

et

al.

-RRB-

It

forms

a

ca.

MDa-complex

with

γ-tubulin

and

depends

on

dynein

for

assembly

onto

centrosomes

-LRB-

Young

et

al.

-RRB-

Pericentrin

plays

a

role

in

recruiting

γ-tubulin

to

centrosomes

in

vertebrate

cells

-LRB-

Dictenberg

et

al.

-RRB-

along

with

several

other

proteins

it

is

part

of

the

centrosomal

targeting

-LRB-

PACT

-RRB-

domain

-LRB-

Gillingham

and

Munro

-RRB-

Mutation

of

the

pericentrin

gene

results

in

loss

of

recruitment

of

several

other

centrosomal

proteins

aside

from

γ-tubulin

which

argues

for

its

essential

role

in

the

centrosome

complex

it

may

provide

a

link

between

centrioles

and

centrosomes

-LRB-

Martinez-Campos

et

al.

-RRB-

Centrins

are

small

proteins

and

members

of

a

highly

conserved

subgroup

of

the

EF-hand

superfamily

of

binding

proteins

that

is

associated

with

centrioles

-LRB-

Bornens

Jurczyk

et

al.

-RRB-

Centrin

is

also

an

intrinsic

component

of

centrosomes

that

has

an

essential

role

in

the

duplication

of

centrosomes

-LRB-

Levy

et

al.

Salisbury

Lutz

et

al.

reviewed

in

Manandhar

et

al.

-RRB-

which

has

been

clearly

determined

in

mutation

experiments

-LRB-

Salisbury

et

al.

-RRB-

NuMA

-LRB-

Nuclear

Mitotic

Apparatus

protein

-RRB-

is

an

essential

cell

cycle-dependent

centrosome-associated

protein

that

plays

a

significant

role

in

the

organization

of

the

mitotic

apparatus

during

mitosis

It

is

a

multifunctional

protein

-LRB-

reviewed

in

Sun

and

Schatten

-RRB-

that

performs

nuclear

functions

as

nuclear

matrix

protein

during

interphase

and

translocates

from

the

nucleus

to

spindle

poles

at

the

onset

of

mitosis

to

form

a

crescent-shaped

complex

around

centrosomes

tethering

microtubules

into

the

accurate

bipolar

organization

-LRB-

addressed

below

in

more

detail

-RRB-

Many

of

the

other

centrosomal

proteins

also

play

significant

roles

in

centrosome

functions

but

are

not

discussed

in

detail

in

this

section

Several

centrosomal

proteins

have

been

implied

in

microtubule

anchoring

to

the

centrosome

including

ninein

that

has

strongly

been

implied

to

serve

as

microtubule

minus-end

anchoring

protein

-LRB-

Mogensen

et

al.

-RRB-

and

dynactin

that

has

a

major

role

in

microtubule

anchorage

at

centrosomes

as

well

as

at

non-centrosomal

anchorage

sites

It

is

preferentially

localized

to

the

mother

centriole

and

plays

a

role

in

microtubule

organization

-LRB-

Quintyne

et

al.

Schroer

Quintyne

and

Schroer

-RRB-

Centrosome

cycle

within

the

cell

cycle

Live

cell

imaging

and

immunofluorescence

microscopy

have

clearly

shown

the

remarkable

shape

changes

of

the

centrosome

which

is

best

visualized

in

reproductive

cells

after

fertilization

requiring

rapid

centrosome

and

microtubule

dynamics

to

position

the

pronuclei

and

prepare

the

zygote

cell

for

mitosis

Figure

shows

stages

of

the

centrosome

cycle

in

a

sea

urchin

egg

which

is

the

model

system

used

by

Boveri

-LRB-

-RRB-

for

many

of

his

centrosome

studies

Centrosomal

material

disperses

around

the

zygote

nucleus

-LRB-

Fig.

-RRB-

to

become

bipolar

in

late

prophase

-LRB-

Fig.

-RRB-

It

becomes

maximally

condensed

during

metaphase

-LRB-

Fig.

-RRB-

and

expands

within

the

anaphase

spindle

poles

-LRB-

Fig.

-RRB-

before

it

becomes

compacted

again

when

telophase

nuclei

form

Figure

d

f

and

h

are

images

of

microtubule

labeling

for

either

the

same

cell

or

cells

of

equivalent

cell

cycle

stages.Fig

structural

cell

cycle-dependent

changes

of

centrosomes

are

shown

here

in

sea

urchin

eggs

during

the

first

cell

cycle

after

fertilization

This

system

had

been

used

by

Theodor

Boveri

for

the

majority

of

his

classic

studies

on

centrosomes

Centrosome

material

disperses

around

the

zygote

nucleus

-LRB-

aarrows

-RRB-

and

separates

to

the

poles

during

prophase

-LRB-

carrows

-RRB-

Centrosomes

become

densely

compacted

in

metaphase

-LRB-

earrows

-RRB-

and

disperse

during

anaphase

-LRB-

garrows

-RRB-

The

correlated

images

for

microtubules

either

from

the

same

cell

or

from

corresponding

cell

cycle

stages

are

shown

in

b

d

f

and

h.

Immunofluorescence

microscopy

of

centrosomes

microtubules

and

DNA

-LRB-

blue

-RRB-

Centrosomes

are

displayed

in

green

microtubules

are

displayed

in

green

-LRB-

b

d

-RRB-

or

red

-LRB-

f

h

-RRB-

Reprinted

with

permission

from

Schatten

et

al.

interphase

a

single

centrosome

is

juxta-positioned

to

the

nucleus

a

c

and

d

show

small

GFP-centrin-labeled

centrosomes

in

mouse

cells

Microtubules

are

detected

with

α-tubulin

and

shown

in

red

b

is

of

an

LNCaP

prostate

cancer

cell

labeled

with

human

autoimmune

antibody

SPJ

displaying

multiple

centrosomal

foci

perhaps

indicating

centrosome

abnormalities

e

shows

a

porcine

fibroblast

cell

labeled

with

γ-tubulin

to

detect

the

centrosome

and

Mitotracker

Rosamine

to

detect

mitochondria

Microtubules

are

shown

in

green

b

reprinted

with

permission

from

Schatten

et

al.

In

interphase

a

single

centrosome

containing

a

pair

of

centrioles

is

juxta-positioned

and

closely

connected

to

the

nucleus

as

shown

in

Fig.

in

an

LNCaP

prostate

cancer

cell

-LRB-

Fig.

-RRB-

mouse

cells

containing

GFP-centrin

label

to

detect

centrosomes

and

labeled

with

α-tubulin

to

detect

microtubules

-LRB-

Fig.

c

d

-RRB-

and

a

pig

fibroblast

cell

labeled

with

γ-tubulin

to

detect

the

centrosome

and

Mitotracker

Rosamine

to

detect

mitochondria

-LRB-

Fig.

-RRB-

Centrosomes

duplicate

shortly

before

the

stage

of

the

cell

cycle

in

a

precisely

orchestrated

program

to

ensure

precise

duplication

into

two

and

not

more

centrosomes

This

process

begins

with

disorientation

of

the

pair

of

centrioles

centriole

duplication

centrosome

disjunction

and

results

in

sister

centrosome

separation

as

reviewed

in

Mack

et

al.

-LRB-

-RRB-

and

Ou

et

al.

-LRB-

-RRB-

While

a

wealth

of

data

has

been

accumulated

on

the

duplication

and

separation

of

centrioles

data

on

the

molecular

events

underlying

centrosome

duplication

disjunction

and

separation

are

only

slowly

accumulating

Centrosome

separation

refers

to

the

spatial

separation

of

centrosome

material

around

the

nucleus

which

is

driven

by

plus

and

minus-end

directed

microtubule

motor

proteins

The

kinase

is

involved

in

centrosome

disjunction

-LRB-

Meraldi

and

Nigg

reviewed

by

Fry

-RRB-

Phosphorylation

of

the

centrosomal

protein

centrin

plays

a

role

in

centrosome

disjunction

at

the

transition

Polo

like

kinases

are

required

for

multiple

stages

of

mitotic

progression

and

have

clearly

been

implicated

in

centrosome

separation

Specific

kinases

have

been

linked

to

centrosome

and

microtubule

dynamics

and

plays

a

critical

role

in

centrosome

and

microtubule

organization

-LRB-

Sun

et

al.

b

c

d

Tong

et

al.

Fan

et

al.

-RRB-

and

both

have

been

implicated

in

microtubule

and

centrosome

functions

in

interphase

and

in

mitosis

-LRB-

Fenton

and

Glover

Donaldson

et

al.

Wang

et

al.

-RRB-

Loss

of

function

has

also

been

associated

with

loss

of

cell

shape

which

indicates

loss

of

microtubule

functions

underneath

the

plasma

membrane

-LRB-

Wang

et

al.

-RRB-

While

the

role

of

centrosomes

in

microtubule

nucleation

and

chromosome

separation

has

been

studied

in

great

detail

centrosomes

have

only

more

recently

been

implicated

in

cytokinesis

Khodjakov

and

Rieder

-LRB-

-RRB-

showed

that

of

cells

with

laser-ablated

centrosomes

failed

to

complete

cytokinesis

perhaps

because

spindles

formed

without

orientation

and

did

not

have

attachment

to

the

cell

cortex

resulting

in

incomplete

chromosome

separation

The

molecular

mechanisms

by

which

centrosomes

exert

an

effect

on

cytokinesis

are

not

clear

but

their

role

as

docking

station

for

cytokinesis-required

regulatory

proteins

is

implied

and

may

include

Plk

and

microtubule

motor

proteins

The

role

of

centrosomes

as

docking

station

where

regulatory

complexes

accumulate

for

cell

cycle

activities

has

only

been

studied

in

recent

years

The

centrosome

serves

as

a

platform

for

a

wide

variety

of

regulatory

molecules

that

play

a

role

in

cell

cycle-specific

functions

but

are

not

themselves

centrosome

proteins

These

complexes

regulate

peripheral

events

through

microtubule-dependent

transport

which

indicates

a

role

of

the

centrosome

as

command

center

that

integrates

signals

using

microtubules

as

signaling

routes

As

such

centrosomes

play

critical

roles

in

cell

cycle

regulation

in

both

mitosis

and

interphase

by

providing

a

central

signaling

station

and

controls

to

trigger

the

next

phases

of

the

cell

cycle

by

integrating

regulating

and

amplifying

signaling

pathways

Phosphorylation

and

posttranslational

modifications

are

other

important

aspects

in

centrosome

regulation

required

for

cell

cycle-specific

changes

which

has

been

reviewed

in

Wilkinson

et

al.

-LRB-

-RRB-

Centrosome

duplication

in

a

regular

cell

cyle

is

typically

well

synchronized

with

the

DNA

cycle

Studies

on

duplication

of

centrosomes

have

been

performed

in

recent

years

to

reveal

that

there

is

a

block

to

reduplication

which

assures

that

centrosomes

are

duplicated

accurately

only

once

within

a

normal

cell

cycle

Elegant

studies

to

determine

the

block

to

reduplication

have

been

performed

by

Wong

and

Stearns

-LRB-

-RRB-

who

fused

phase

cells

containing

one

centrosome

with

phase

cells

containing

two

centrosomes

to

determine

if

an

already

duplicated

centrosome

would

re-duplicate

under

these

conditions

Interestingly

the

centrosome

duplicated

while

the

centrosome

did

not

which

led

to

the

conclusion

that

there

is

an

intrinsic

block

to

reduplication

within

the

centrosome

Initiation

of

centrosome

duplication

is

under

cytoplasmic

control

and

driven

by

cyclin-dependent

kinase

-LRB-

-RRB-

complexed

with

cyclin

E

or

cyclin

A

that

rises

during

the

late

stage

-LRB-

reviewed

in

Sluder

-RRB-

These

results

were

compiled

by

several

laboratories

in

the

late

using

Xenopus

eggs

-LRB-

Hinchcliffe

et

al.

Lacey

et

al.

-RRB-

and

mammalian

somatic

cells

-LRB-

Mantel

et

al.

Matsumoto

et

al.

-RRB-

It

was

later

shown

that

initiation

of

centrosome

duplication

also

requires

calcium/calmodulin-dependent

kinase

II

-LRB-

CaMKII

-RRB-

-LRB-

Matsumoto

and

Maller

-RRB-

CaMKII

phosphorylates

centrosome

proteins

in

vitro

-LRB-

Pietromonaco

et

al.

-RRB-

and

is

localized

to

spindle

poles

-LRB-

Ohta

et

al.

-RRB-

Ubiquitin-mediated

proteolysis

of

centrosomal

proteins

may

be

involved

in

the

block

to

reduplication

as

proposed

by

several

investigators

-LRB-

Tugendreich

et

al.

Freed

et

al.

Gstaiger

et

al.

-RRB-

who

showed

localization

of

a

variety

of

components

of

the

SFC

proteolysis

pathway

as

well

as

the

proteasome

to

centrosomes

in

human

cells

Centrosome

duplication

and

DNA

replication

both

require

hyperphosphorylation

of

the

retinoblastoma

-LRB-

RB

-RRB-

protein

and

activation

of

Although

the

centrosome

cycle

and

DNA

cycle

are

normally

coupled

through

cell

cycle-dependent

checkpoints

it

has

been

possible

to

dissociate

the

centrosome

cycle

from

other

cell

cycle

events

Application

of

cycloheximide

to

Xenopus

oocytes

has

resulted

in

centrosome

duplication

while

other

cell

cycle

events

were

inhibited

-LRB-

Gard

et

al.

-RRB-

Hydroxyurea

or

aphidicolin

also

resulted

in

continuation

of

the

centrosome

cycle

in

CHO

cells

producing

supernumerary

centrosomes

while

DNA

replication

was

inhibited

-LRB-

Balczon

et

al.

-RRB-

Normally

the

DNA

cycle

and

the

centrosome

cycle

are

intimately

coupled

and

synchronized

in

a

regular

cell

cycle

When

the

nucleus

or

DNA

is

damaged

centrosomes

become

inactivated

in

a

well-orchestrated

process

The

γ-TuRC

dissociates

from

centrosomes

the

spindle

fails

to

assemble

resulting

in

failure

of

chromosome

segregation

Figure

is

a

schematic

representation

of

a

typical

centrosome

cycle

within

a

typical

cell

cycle

Please

see

figure

legend

for

description.Fig

typical

mammalian

centrosome

cycle

within

the

cell

cycle

The

single

interphase

centrosome

-LRB-

a

-RRB-

is

closely

associated

with

the

nucleus

and

nucleates

an

array

of

interphase

microtubules

Centrosome

duplication

occurs

during

the

S

phase

in

synchrony

with

DNA

dupliction

-LRB-

b

-RRB-

Centrosome

separation

of

the

duplicated

centrosome

toward

opposite

poles

takes

place

in

the

early

prophase

stage

-LRB-

c

-RRB-

The

bipolar

mitotic

apparatus

becomes

established

when

centrosomes

have

reached

the

opposite

poles

and

the

nuclear

envelope

has

broken

down

-LRB-

d

-RRB-

During

this

stage

interphase

centrosomes

mature

into

mitotic

centrosomes

acquiring

mitosis-associated

centrosomal

proteins

including

NuMA

that

moves

out

of

the

nucleus

to

become

a

mitotic

centrosome-associated

protein

The

metaphase

centrosome

-LRB-

e

-RRB-

becomes

highly

compacted

to

organize

the

metaphase

spindle

with

microtubules

attached

to

the

kinetochores

Anaphase

is

the

stage

when

centrosomal

material

becomes

decompacted

again

-LRB-

f

-RRB-

before

reorganizing

into

interphase

centrosomes

that

associate

with

the

nuclei

of

the

separating

daughter

cells

-LRB-

g

-RRB-

Centrosomes

shaded

yellow

with

centrioles

and

PCM

displayed

in

black

nuclei

shaded

orange

microtubules

displayed

as

black

rods

chromosomes

displayed

in

black

Modified

from

Sun

and

Schatten

Centrosome

maturation

into

mitotic

centrosomes

One

of

the

most

important

functions

of

the

centrosome

is

its

role

in

establishing

the

bipolar

mitotic

spindles

that

orchestrate

the

precisely

balanced

segregation

of

chromosomes

To

accomplish

this

important

function

a

major

reorganization

of

centrosomal

material

occurs

at

the

transition

in

vertebrate

cells

which

is

referred

to

as

centrosome

maturation

During

this

time

some

centrosomal

proteins

become

diminished

while

others

such

as

the

γ-TuRC

become

enriched

-LRB-

Khodjakov

and

Rieder

-RRB-

and

γ-tubulin

increases

three

to

five-fold

-LRB-

Khodjakov

and

Rieder

-RRB-

Cyclin-dependent

kinases

play

a

significant

role

in

centrosome

cycle

progression

The

transition

requires

B

as

well

as

A

which

has

been

reviewed

in

detail

by

Fry

and

Hames

-LRB-

-RRB-

is

localized

to

centrosomes

at

the

onset

of

mitosis

-LRB-

Bailly

et

al.

Pockwinse

et

al.

-RRB-

and

it

has

been

shown

that

B

activation

is

detected

in

centrosomes

during

prophase

-LRB-

Jackman

et

al.

-RRB-

The

centrosome

cycle

is

tied

to

the

centriole

cycle

and

both

are

closely

coupled

with

the

DNA

cycle

At

the

transition

from

the

to

S

phase

centriole

duplication

begins

and

continues

through

when

the

replicating

pro-centriole

elongates

Two

duplicated

juxta-positioned

centrioles

are

clearly

apparent

at

the

stage

The

two

centrosome

complexes

are

held

to

each

other

throughout

interphase

and

undergo

disjunction

prior

to

entry

into

mitosis

-LRB-

Mayor

et

al.

-RRB-

Centrosome

maturation

takes

place

by

acquiring

mitotic

centrosome

proteins

including

-LRB-

Golsteyn

et

al.

-RRB-

NuMA

-LRB-

Merdes

et

al.

-RRB-

and

others

while

interphase

centrosome

proteins

such

as

-LRB-

Fry

et

al.

-RRB-

or

Nlp

-LRB-

Casenghi

et

al.

-RRB-

are

removed

Increased

recruitment

of

γ-TuRC

to

the

centrosome

assures

increased

nucleation

of

microtubules

for

spindle

formation

While

centriole

separation

has

been

studied

in

great

detail

in

recent

years

our

knowledge

about

centrosome

separation

is

still

incomplete

and

we

do

not

yet

fully

understand

how

centrosomes

are

separated

into

two

Separation

is

complete

when

cells

re-enter

with

a

single

centrosome

surrounding

a

pair

of

centrioles

One

of

the

well-studied

essential

mitotic

centrosome-associated

proteins

is

NuMA

After

nuclear

envelope

breakdown

-LRB-

NEB

-RRB-

NuMA

becomes

a

most

significant

centrosome-associated

protein

-LRB-

reviewed

in

Sun

and

Schatten

-RRB-

NuMA

does

not

associate

with

interphase

centrosomes

It

serves

as

nuclear

matrix

protein

in

the

interphase

nucleus

and

becomes

dispersed

into

the

cytoplasm

during

NEB

a

process

in

which

cyclin

B

plays

a

critical

role

B

dependent

phosphorylation

is

important

for

translocation

of

NuMA

from

the

nucleus

into

the

cytoplasm

-LRB-

Saredi

et

al.

and

references

therewithin

-RRB-

where

it

associates

with

microtubules

using

a

dynein

dynactin-mediated

mechanism

to

be

translocated

along

microtubules

to

the

centrosome

area

Next

to

become

a

functional

mitotic

protein

NuMA

needs

to

be

translocated

from

the

cytoplasm

to

the

mitotic

centrosomes

In

this

highly

regulated

process

microtubules

and

dynein

dynactin

play

crucial

roles

in

transporting

NuMA

to

the

mitotic

poles

where

it

forms

an

insoluble

crescent

around

centrosomes

that

tethers

microtubules

precisely

into

the

bipolar

configuration

that

forms

the

mitotic

apparatus

-LRB-

Merdes

and

Cleveland

-RRB-

NuMA

is

distributed

to

the

separating

centrosomes

during

early

mitosis

It

ensures

minus-end

binding

and

stabilization

of

microtubules

on

the

centrosome

area

that

faces

chromosomes

NuMA

facilitates

cross-linking

of

spindle

microtubules

aiding

in

the

organization

and

stabilization

of

spindle

poles

from

early

mitosis

to

anaphase

To

exit

its

mitotic

functions

and

to

relocate

to

the

nucleus

NuMA

needs

to

be

dissociated

from

the

mitotic

spindle

poles

through

a

process

that

requires

B

-LRB-

Gehmlich

et

al.

-RRB-

Destruction

of

cyclin

B

promotes

exit

from

mitosis

which

starts

at

the

mitotic

poles

and

is

linked

to

disassembling

the

mitotic

centrosome

complex

Failure

of

NuMA

to

relocate

to

the

nucleus

will

result

in

cytoplasmic

NuMA

spots

that

organize

abnormal

microtubule

formations

-LRB-

Gehmlich

et

al.

-RRB-

As

nuclear

matrix

protein

NuMA

is

involved

in

DNA

replication

and

transcription

It

displays

distinct

staining

patterns

in

the

nucleus

-LRB-

Merdes

and

Cleveland

Zeng

Gobert

et

al.

-RRB-

and

stains

heavily

in

mitosis

when

using

anti-NuMA

antibodies

-LRB-

Saredi

et

al.

Merdes

et

al.

Gobert

et

al.

Zhong

et

al.

Liu

et

al.

-RRB-

Inaccurate

NuMA

regulation

and

atypical

NuMA

distribution

will

result

in

abnormal

mitosis

as

has

been

reported

for

cancer

cells

-LRB-

Saunders

et

al.

-RRB-

Some

of

the

important

mitotic

cell

cycle

regulators

are

concentrated

at

the

centrosome

and

include

Polo

and

Aurora

A

kinases

-LRB-

Barr

and

Gergely

-RRB-

and

B

kinase

-LRB-

Jackman

et

al.

-RRB-

Some

of

these

proteins

bind

to

the

anaphase

promotion

complex/cyclosome

-LRB-

APC/C

-RRB-

The

activated

degrades

cyclin

B

and

securin

to

allow

cell

cycle

exit

from

mitosis

-LRB-

Kramer

et

al.

Huang

and

Raff

Wakefield

et

al.

-RRB-

Microtubule

motor

proteins

are

essential

for

the

composition

of

a

functional

mitotic

centrosome

as

many

centrosome

proteins

are

shuttled

along

microtubules

to

the

centrosome

core

structure

including

pericentrin

and

NuMA

Pericentriolar

satellites

-LRB-

or

granules

-RRB-

may

function

as

cargo

vehicle

for

various

proteins

including

ninein

centrin

and

pericentrin

-LRB-

Dammermann

and

Merdes

-RRB-

These

granules

move

along

microtubules

in

a

dynein-dependent

process

-LRB-

Kubo

et

al.

Kubo

and

Tsukita

-RRB-

The

microtubule

motor

proteins

dynein

and

kinesin

are

crucial

for

transport

of

cargo

along

microtubules

and

balanced

transport

is

important

to

maintain

cellular

homeostasis

In

most

cells

transport

of

cellular

organelles

such

as

mitochondria

and

vesicles

along

microtubules

is

mediated

by

the

kinesin

family

of

motor

proteins

to

the

plus

end

of

microtubules

and

by

cytoplasmic

dyneins

and

its

co-factor

dynactin

to

the

minus

ends

-LRB-

reviewed

by

Welte

-RRB-

Bidirectional

transport

of

cell

organelles

and

vesicles

is

also

possible

-LRB-

Haggeness

et

al.

-RRB-

Imbalanced

transport

can

lead

to

pathologies

related

to

centrosome

and

microtubule

functions

as

well

as

to

failures

in

organelle

and

vesicle

distribution

Centrosome

and

microtubule

pathologies

are

the

result

of

disrupted

transport

Disruption

of

transport

is

the

cause

or

effect

in

a

chain

of

signal

transduction

events

disruption

depletes

the

resources

of

proteins

and

generates

signals

regulating

transcription

Following

disruption

is

a

cascade

of

declines

resulting

in

secondary

pathologies

Microtubule-based

transport

processes

are

vital

for

cell

functions

and

dysfunctional

transport

along

microtubules

has

been

implicated

in

cellular

deterioration

and

disease

such

as

Alzheimer

's

As

is

the

case

for

transport

toward

the

minus

end

transport

of

microtubules

toward

centrosomes

dynein

is

involved

in

the

mediation

of

transloction

along

microtubules

to

the

plus

ends

towards

the

cell

periphery

Dynein

motors

may

be

linked

to

actin

or

intermediate

filaments

as

has

been

shown

for

neurons

in

which

dynein

and

dynactin

are

responsible

for

translocation

of

microtubules

from

the

centrosome

in

the

cell

body

into

the

axon

-LRB-

Baas

Ahmad

et

al.

-RRB-

Myosin

may

play

a

role

in

connecting

microtubules

to

actin

filaments

-LRB-

Cao

et

al.

-RRB-

Supernumerary

centrosomes

role

in

genomic

instability

and

cancer

Boveri

's

visionary

book

on

centrosome

abnormalities

in

cancer

had

originally

been

translated

from

German

into

English

as

its

significance

and

powerful

implications

had

been

recognized

from

the

onset

-LRB-

Boveri

-RRB-

This

book

has

recently

been

re-edited

again

because

of

its

significance

that

continues

to

fascinate

investigators

in

the

centrosome

field

as

well

as

others

The

visionary

research

and

intellectually

remarkable

interpretations

implied

one

cell

with

abnormal

centrosomes

as

the

primordial

tumor

cell

Boveri

's

observations

of

dispermic

eggs

developing

multipolar

mitoses

and

his

brilliant

thought

process

to

imply

supernumerary

centrosomes

in

tumor

has

found

enormous

recognition

during

the

past

decade

when

several

groups

followed

up

on

Boveri

's

initial

findings

and

analyzed

archived

tumor

tissues

of

various

types

Since

then

multipolar

mitoses

have

been

identified

in

a

large

number

of

cancers

as

hallmarks

of

tumor

tissue

with

direct

implications

in

the

causes

for

aneuploidy

-LRB-

Lingle

and

Salisbury

Lingle

et

al.

Pihan

et

al.

Pihan

and

Doxsey

Schatten

et

al.

b

b

b

c

Brinkley

and

Goepfert

and

others

reviewed

in

Goepfert

and

Brinkley

-RRB-

Moreover

overexpression

of

specific

centrosome

proteins

resulted

in

abnormal

centrosome

configurations

and

aneuploidy

-LRB-

Lingle

et

al.

Katayama

et

al.

-RRB-

confirming

the

important

role

of

centrosomes

in

cancer

development

and

progression

Increased

centrosome

number

and

volume

supernumerary

centrioles

accumulation

of

increased

PCM

and

abnormal

phosphorylation

of

centrosomes

have

all

been

associated

with

cancer

cell

centrosomes

followed

by

loss

of

cell

polarity

-LRB-

Lingle

et

al.

Schatten

et

al.

-RRB-

Deregulation

of

centrosome

duplication

and

genes

implicated

in

centrosome

amplification

are

processes

that

lead

to

cascades

of

cell

cycle-related

abnormalities

The

loss

of

is

associated

with

multiple

cycles

of

centrosome

duplication

in

one

S

phase

resulting

in

multiple

centrosome

numbers

-LRB-

Pihan

and

Doxsey

-RRB-

might

play

a

role

in

the

block

to

reduplication

in

synchrony

with

the

DNA

cycle

Viral

oncoproteins

that

inactivate

also

result

in

cells

with

supernumerary

centrosomes

as

has

been

shown

for

the

papilloma

virus

-LRB-

reviewed

in

Münger

and

Duensing

-RRB-

However

different

investigators

have

challenged

that

loss

of

directly

affects

centrosome

duplication

-LRB-

Meraldi

et

al.

-RRB-

and

attribute

centrosome

abnormalities

to

missing

checkpoint

functions

after

loss

of

The

breast

cancer

suppressor

gene

may

also

play

a

role

in

deregulation

of

centrosome

duplication

as

targeted

deletion

of

leads

to

centrosome

amplification

-LRB-

Xu

et

al.

-RRB-

However

the

mechanism

by

which

affects

centrosome

duplication

remains

to

be

fully

investigated

plays

a

role

in

checkpoint

functions

which

may

result

in

loss

of

the

block

to

centrosome

reduplication

Aurora

kinases

play

central

roles

in

the

mitotic

process

and

cell

division

and

Aurora

A

has

been

implicated

in

centrosome

amplification

in

breast

cancer

Aurora

A

localizes

to

centrosomes

and

overexpression

of

Aurora

A

causes

multipolar

mitotic

spindles

and

is

implicated

in

early

development

of

mammary

tumors

Oncogenic

insults

may

result

in

a

high

number

of

mutation

rates

in

cells

predispositioned

to

tumor

and

the

mutation

rate

increases

in

cells

that

have

reached

replicative

senescence

In

contrast

cervical

carcinogenesis

has

strongly

been

associated

with

infections

by

high-risk

human

papillomavirus

-LRB-

HPVs

-RRB-

It

has

been

suggested

that

the

HPV

oncoprotein

may

induce

primary

centrosome

duplication

errors

and

act

as

mitotic

mutator

-LRB-

reviewed

in

Münger

and

Duensing

-RRB-

As

with

other

cancers

abnormal

multipolar

mitoses

resulting

from

supernumerary

centrosomes

have

clearly

been

associated

with

HPV-associated

lesions

and

centrosome

abnormalities

are

already

detected

in

early

stages

of

tumor

development

Studies

have

shown

that

uncoupling

of

the

cell

division

cycle

from

the

centrosome

cycle

subverts

centrosome

homeostasis

-LRB-

Duensing

et

al.

-RRB-

An

emerging

field

in

centrosome

research

is

their

role

in

aging

Supernumerary

centrosomes

are

thought

to

be

associated

with

aging

and

have

been

shown

in

senescing

cells

-LRB-

Schatten

et

al.

-RRB-

Many

of

the

cell

cycle

regulators

that

play

a

role

at

the

transition

from

to

M

phases

are

downregulated

in

aging

cells

-LRB-

Ly

et

al.

-RRB-

which

is

the

most

significant

phase

for

reorganizations

of

microtubules

and

centrosomes

As

mentioned

above

centrosomes

undergo

major

changes

in

phosphorylation

at

the

transition

and

it

has

been

shown

that

centrosomes

in

aging

cells

have

lower

activity

in

centrosome-associated

protein

kinases

-LRB-

Cande

Huang

-RRB-

Plk

has

been

identified

as

one

of

the

gene

products

that

are

down-regulated

in

aging

cells

-LRB-

Ly

et

al.

-RRB-

which

will

have

significant

consequences

for

centrosome

and

microtubule

organization

This

is

important

considering

that

many

diseases

of

aging

involve

inaccurate

microtubule

organization

coupled

with

transport

dysfunctions

Centrosome-independent

pathways

and

non-centrosomal

microtubule

arrays

Typical

centrosomes

are

absent

in

most

plant

cells

and

centrosome

material

without

centrioles

compose

the

meiotic

spindle

in

animal

oocyte

cells

Somatic

cells

also

can

assemble

bipolar

spindles

in

the

absence

of

typical

centrosomes

-LRB-

Meraldi

and

Nigg

Manandhar

et

al.

-RRB-

In

animal

cells

microsurgery

to

remove

the

centrosome

from

transformed

mouse

fibroblast

cells

-LRB-

cells

-RRB-

resulted

in

reorganization

of

microtubules

without

a

centrosome

but

absence

of

a

mitotic

spindle

and

cell

cycle

progression

arrest

implying

that

the

centrosome

is

important

for

cell

cycle

progression

Although

the

centrosome

is

vital

for

animal

cells

and

there

are

no

viable

mutants

in

animal

cells

that

lack

centrosomes

there

are

centrosome-independent

pathways

that

have

recently

been

explored

by

destroying

centrosomes

in

situ

with

focused

pulses

of

high-energy

-LRB-

laser

-RRB-

light

termed

the

ablative

photo-decomposition

approach

-LRB-

reviewed

in

Khodjakov

and

Rieder

-RRB-

These

studies

used

Green

Fluorescent

Protein

-LRB-

GFP

-RRB-

fusion

for

detection

of

centrosomes

for

precise

centrosome

targeting

and

to

follow

the

extent

of

centrosome

destruction

in

live

cells

These

studies

showed

that

some

acentrosomal

spindle

formation

is

possible

when

centrosomes

are

destroyed

In

parthenogenetic

human

rhesus

and

bovine

embryos

a

bipolar

spindle

is

formed

at

the

time

of

first

mitosis

without

apparent

γ-tubulin

and

pericentrin

foci

at

the

spindle

poles

which

argues

that

mitotic

spindles

can

self-assemble

without

functional

MTOCs

Acentrosomal

meiotic

and

mitotic

spindles

have

been

described

-LRB-

Lee

et

al.

Megraw

et

al.

Bonaccorsi

et

al.

Khodjakov

et

al.

Khodjakov

and

Rieder

Raff

-RRB-

It

is

not

entirely

clear

how

the

spindle

poles

are

formed

but

microtubule

motor

activities

have

been

implicated

in

this

process

-LRB-

Walczak

et

al.

-RRB-

Release

of

entire

microtubules

from

the

centrosome

complex

has

been

well

documented

as

the

main

mechanism

to

produce

non-centrosomal

free

microtubules

in

neurons

-LRB-

Ahmad

et

al.

-RRB-

The

microtubule-severing

protein

katanin

has

been

implicated

in

this

process

-LRB-

Ahmad

et

al.

Baas

McNally

and

Vale

McNally

et

al.

-RRB-

Katanin

may

also

play

a

role

in

M-phase

severing

processes

at

the

spindle

poles

in

mammalian

cells

when

spindle

pole

microtubules

become

depolymerized

-LRB-

McNally

et

al.

McNally

and

Thomas

-RRB-

Katanin

also

plays

a

role

in

severing

of

axonemal

microtubules

-LRB-

Lohret

et

al.

-RRB-

and

katanin-like

proteins

have

been

identified

in

plants

-LRB-

Burk

et

al.

-RRB-

Non-centrosomal

microtubule

arrays

have

been

described

for

polarized

epithelial

cells

-LRB-

reviewed

by

Morgensen

-RRB-

to

serve

various

specialized

functions

of

differentiated

cells

In

these

cells

centrosomes

are

present

but

organize

fewer

microtubules

while

most

of

the

cellular

microtubules

are

non-centrosomal

and

the

minus

ends

are

localized

toward

the

plasma

membrane

without

γ-tubulin

anchorage

Such

organization

is

seen

in

rat

Sertoli

cells

-LRB-

Vogl

et

al.

-RRB-

Other

examples

of

non-centrosomal

microtubules

with

significant

cellular

functions

are

found

in

the

organ

of

Corti

in

the

inner

ear

and

during

vertebrate

myogenesis

The

microtubule

minus

end-anchoring

protein

ninein

is

localized

to

the

non-centrosomal

microtubule

minus-end

sites

in

MDCK

polarized

epithelial

cells

while

γ-tubulin

or

pericentrin

are

absent

Microtubule-associated

proteins

-LRB-

MAPs

-RRB-

at

the

minus

ends

may

play

a

role

in

minus

end-capping

of

microtubules

preventing

their

depolymerization

Plus-end

capping

involves

Rho

GTPases

and

the

downstream

effector

mDia

-LRB-

mouse

diaphanous-related

formins

-RRB-

-LRB-

Gundersen

Palazzo

et

al.

-RRB-

Other

plus-end

capping

candidates

are

dynein/dynactin

and

APC

-LRB-

adenomatis

polyposis

coli

-RRB-

protein

-LRB-

reviewed

in

Mogensen

-RRB-

It

is

thought

that

these

non-centrosomal

microtubules

originate

from

centrosomes

Dynactin

also

localizes

to

the

non-centrosomal

microtubule

minus-end

sites

in

MDCK

polarized

epithelial

cells

in

further

support

for

anchorage

sites

at

the

cell

apex

A

microtubule

release

and

capture

mechanism

has

been

implicated

in

the

generation

of

non-centrosomal

apico-basal

microtubule

formations

in

polarized

epithelial

cells

The

non-centrosomal

microtubule

organization

provides

support

for

the

idea

that

two

functionally

distinct

microtubule

minus-end-associated

complexes

are

independent

from

each

other

and

comprise

the

nucleating

complex

and

the

anchoring

complex

The

role

of

ninein

in

microtubule

anchorage

at

the

centrosomal

PCM

has

been

reported

by

Mogensen

et

al.

-LRB-

-RRB-

The

minus

end

microtubule

anchoring

capabilities

by

ninein

have

been

demonstrated

by

overexpression

of

ninein

causing

an

increase

of

microtubule

anchorage

and

decrease

in

microtubule

release

The

loss

of

microtubule

radial

organization

has

been

demonstrated

by

using

either

anti-ninein

antibodies

or

RNAi

depletion

of

ninein

-LRB-

Dammermann

and

Merdes

-RRB-

Centrosome

abnormalities

and

mitotic

cell

death

Alterations

in

centrosome

organization

and

function

are

associated

with

cellular

stress

and

may

be

part

of

the

mitotic

catastrophe

response

characteristic

for

programmed

mitotic

cell

death

Centrosome

structure

and

function

are

directly

affected

by

chaotropic

agents

such

as

formamide

-LRB-

Schatten

et

al.

-RRB-

and

in

response

to

heat

shock

and

DNA

damage

While

mild

heatshock

treatment

triggers

repair

mechanisms

and

centrosome

thermotolerance

severe

heat

shock

leads

to

cell

death

by

a

mitotic

catastrophe

mechanism

Formamide

induces

the

formation

of

multipolar

spindles

perhaps

as

a

result

of

centrosomal

protein

denaturation

-LRB-

Schatten

et

al.

-RRB-

Tripolar

and

fractionated

centrosomes

after

treatment

of

cells

with

formamide

are

shown

in

Fig.

and

b

respectively

The

effects

of

heat

shock

and

other

environmental

stresses

have

been

studied

in

various

cell

systems

that

all

displayed

centrosome

alterations

was

localized

to

centrosomes

in

HeLa

cells

-LRB-

Rattner

-RRB-

Nakahata

et

al.

-LRB-

-RRB-

reported

centrosomal

dysfunction

and

non-apoptotic

mitotic

catastrophe

in

human

tumor

cells

which

has

also

been

reported

for

cells

exposed

to

radiation

-LRB-

reviewed

in

Sato

et

al.

-RRB-

Some

of

the

causes

for

mitotic

catastrophe

are

related

to

checkpoint

or

DNA

repair

failures

which

is

thought

to

be

a

genetically

programmed

response

to

genotoxic

stress

leading

to

centrosome

inactivation

-LRB-

Sibon

et

al.

-RRB-

Loss

of

γ-tubulin

loss

of

pericentrin

and

multipolar

spindles

as

well

as

additional

PCM

foci

and

structural

alterations

are

some

of

the

characteristics

associated

with

heat

shock

-LRB-

Vidair

et

al.

Barrau

et

al.

Knox

et

al.

Debec

et

al.

Marcaillou

et

al.

Debec

and

Marcaillou

-RRB-

Cells

with

mitotic

catastrophe

and

multipolar

spindles

are

eliminated

from

a

cell

population

by

a

process

resulting

in

cell

death

which

has

been

reported

for

mammalian

somatic

cells

as

well

as

for

Drosophila

cells

in

culture

-LRB-

Schatten

et

al.

-RRB-

This

process

is

used

in

addition

to

apoptosis

to

eliminate

cells

with

dysfunctional

mitotic

cells

from

a

normal

cell

population

It

has

also

been

shown

to

account

for

causes

of

embryo

abnormalities

after

somatic

cell

nuclear

transfer

-LRB-

SCNT

-RRB-

-LRB-

reviewed

in

Schatten

et

al.

Zhong

et

al.

-RRB-

Such

cells

undergoing

mitotic

cell

death

are

negative

for

TUNEL

staining

and

isolated

DNA

is

not

characterized

by

laddering

that

is

typical

for

apoptosis

-LRB-

Roninson

et

al.

Nabha

et

al.

-RRB-

Apoptotic

inhibitors

are

not

effective

in

these

cells

and

apoptotic

bodies

are

not

formed

while

fragmentation

of

centrosomes

appears

characteristic

for

mitotic

cell

death.Fig

chaotropic

agent

formamide

causes

centrosomal

damage

and

results

in

tripolar

-LRB-

a

-RRB-

or

multiple

-LRB-

b

-RRB-

centrosomal

foci

as

shown

here

in

an

example

using

sea

urchin

eggs

as

model

system

Centrosomes

labeled

red

-LRB-

a

-RRB-

or

green

-LRB-

b

-RRB-

Microtubules

in

b

labeled

red

a

modified

from

Schatten

et

al.

Ionizing

radiation

is

well

known

to

induce

aberrations

in

centrosome

number

in

tumor

cell

lines

but

the

underlying

causes

are

not

well

understood

-LRB-

Sato

et

al.

b

-RRB-

As

discussed

above

for

heat

shock

damage

the

DNA

checkpoint

pathways

may

play

a

role

in

radiation-induced

centrosome

aberrations

or

it

may

directly

affect

centrosome

structure

Mitotic

cell

death

is

seen

in

most

cell

types

after

ionizing

radiation

while

apoptosis

is

primarily

seen

in

lymphoma

and

leukemia

cells

Exposure

to

gamma-irradiation

at

a

single

dose

of

Gy

resulted

in

increased

numbers

of

centrosomes

with

varied

abnormalities

and

cell

fractionation

-LRB-

reviewed

in

Sato

et

al.

-RRB-

Mitotic

cell

death

after

mitotic

catastrophe

is

also

caused

by

other

chemotherapeutic

agents

such

as

doxorubicin

cisplatin

bleomycin

and

taxol

-LRB-

Roninson

et

al.

Schatten

et

al.

b

-RRB-

The

causes

for

centrosome

number

increases

after

irradiation

are

not

clear

but

loss

of

cell

cycle

regulators

such

as

and

its

downstream

targets

and

have

been

implicated

in

supernumerary

centrosomes

-LRB-

Fukasawa

et

al.

Hollander

et

al.

Mantel

et

al.

Carroll

et

al.

-RRB-

Dissociation

of

centrosome

cycles

from

DNA

cycles

may

be

among

the

causes

for

centrosome

aberrations

and

multipolar

spindle

formations

after

irradiation

Aberrant

hypermethylation

has

recently

been

implicated

in

inactivation

of

checkpoint

genes

that

may

influence

cell

cycle-dependent

centrosome

abnormalities

as

reported

for

pancreatic

cancer

-LRB-

Ohki

et

al.

Sato

et

al.

-RRB-

Environmental

stress

can

result

in

the

formation

of

aggresomes

which

are

oftentimes

localized

close

to

centrosomes

and

are

thought

to

be

the

result

of

misfolded

proteins

-LRB-

Ellgaard

et

al.

Johnston

et

al.

Wojcik

and

DeMartino

Kopito

Roth

et

al.

-RRB-

Some

of

the

aggresomes

contain

γ-tubulin

and

are

associated

with

disease

or

neurodegenerative

disorders

such

as

Parkinson

's

disease

and

dementia

-LRB-

McNaught

et

al.

-RRB-

Centrosomes

in

reproduction

including

fertilization

and

somatic

cell

nuclear

transfer

-LRB-

SCNT

-RRB-

Boveri

's

brilliant

discoveries

on

centrosomes

primarily

came

from

his

studies

on

centrosomes

during

fertilization

and

cell

division

in

sea

urchin

eggs

-LRB-

Boveri

-RRB-

which

resulted

in

a

wealth

of

profound

information

on

the

important

contribution

of

sperm

centrosomes

for

successful

fertilization

His

work

on

reproduction

also

resulted

in

the

remarkable

insights

that

supernumerary

centrosomes

are

at

the

core

of

malignant

tumors

-LRB-

Boveri

-RRB-

which

was

based

on

his

observations

that

dispermic

eggs

-LRB-

eggs

fertilized

with

two

sperm

-RRB-

developed

multipolar

mitotic

poles

An

example

of

a

dispermic

sea

urchin

egg

is

seen

in

Fig.

Boveri

had

recognized

that

sperm

contribute

the

dominant

centrosomal

material

that

was

detected

by

staining

with

iron

hematoxylin

and

could

be

traced

throughout

cell

division

and

development

In

more

recent

years

staining

of

centrosomes

with

immunological

probes

confirmed

that

in

most

animal

species

except

for

the

mouse

-LRB-

Schatten

et

al.

-RRB-

dominant

centrosomal

material

is

contributed

by

sperm

-LRB-

reviewed

in

Manandhar

et

al.

Sun

and

Schatten

-RRB-

and

biparental

centrosome

contributions

to

the

zygote

are

typical

for

most

species

More

detailed

studies

using

specific

immunofluorescent

probes

to

specific

centrosome

proteins

provided

additional

insights

into

centrosomal

contributions

during

gametogenesis

and

fertilization

-LRB-

reviewed

by

Manandhar

et

al.

-RRB-

These

studies

showed

that

the

sperm

retains

its

proximal

centriole

while

losing

most

of

the

PCM

The

oocyte

on

the

other

hand

degenerates

centrioles

while

retaining

centrosomal

proteins

The

sperm

's

proximal

centriole

recruits

egg

proteins

shortly

after

sperm

incorporation

including

γ-tubulin

centrin

pericentrin

and

NuMA

to

the

sperm

centriolar

complex

The

recruitment

of

maternal

γ-tubulin

to

the

sperm

's

centrosomal

γ-tubulin

results

in

a

significant

accumulation

of

γ-tubulin

The

blended

centrosome

after

fertilization

also

contains

centrin

from

both

sperm

and

egg

The

zygotic

centrosome

that

is

closely

associated

with

the

decondensing

male

pronucleus

then

organizes

a

radial

aster

that

grows

toward

the

female

pronucleus

The

centriole-centrosome

complex

duplicates

during

the

pronuclear

stage

A

zygote

aster

forms

around

the

appositioned

-LRB-

or

fused

-RRB-

male

and

female

pronuclei

-LRB-

zygote

nucleus

-RRB-

and

centrosomes

separate

to

form

the

bipolar

mitotic

apparatus

in

preparation

for

cell

division

-LRB-

reviewed

in

Sun

and

Schatten

-RRB-

Dysfunctional

sperm

centrosomes

dysfunctional

zygote

centrosomes

and

polyspermy

have

been

implicated

in

causes

for

male-derived

infertility

-LRB-

reviewed

in

Schatten

-RRB-

while

aging

of

oocyte

centrosomes

is

among

the

major

causes

for

infertility

in

older

women

-LRB-

Battaglia

et

al.

-RRB-

as

dysfunctional

centrosomes

are

implied

in

the

causes

for

aneuploidy

It

is

estimated

that

the

rates

of

aneuploidy

in

preimplantation

human

embryos

is

as

high

as

-LRB-

Munne

and

Cohen

-RRB-

Fig.

tetrapolar

spindle

displaying

microtubules

-LRB-

green

-RRB-

and

chromosomes

-LRB-

red

-RRB-

as

a

result

of

dispermy

in

a

sea

urchin

egg

Such

results

on

dispermic

eggs

had

stimulated

Boveri

to

propose

that

centrosome

abnormalities

are

at

the

core

of

malignant

tumors

Image

produced

in

collaboration

at

the

Integrated

Microscopy

Resource

at

the

University

of

Wisconsin

Centrosome

remodeling

after

nuclear

transfer

communication

between

embryonic

and

somatic

cells

Cloning

of

animals

by

somatic

cell

nuclear

transfer

-LRB-

SCNT

-RRB-

involves

cellular

communication

of

embryonic

cells

with

somatic

cell

nuclei

and

somatic

cell

centrosomes

from

donor

cells

In

recent

years

cloning

of

animals

by

SCNT

has

opened

up

new

avenues

to

produce

genetically

modified

species

with

higher

nutritional

value

or

improved

traits

that

are

useful

in

agriculture

Genetically

modified

pigs

have

been

produced

as

models

for

human

disease

and

for

various

other

biomedical

applications

-LRB-

reviewed

in

Prather

Schatten

et

al.

-RRB-

However

one

of

the

major

difficulties

to

overcome

in

cloning

is

the

low

cloning

efficiency

that

ranges

from

to

in

different

animals

Centrosomal

dysfunctions

have

been

identified

as

possible

causes

for

low

cloning

efficiencies

in

several

species

including

the

pig

-LRB-

Zhong

et

al.

-RRB-

and

rhesus

monkeys

-LRB-

Simerly

et

al.

-RRB-

and

centrosomal

dysfunctions

may

also

play

a

role

in

developmental

abnormalities

that

are

frequently

associated

with

cloning

SCNT

involves

transferring

a

somatic

cell

nucleus

with

known

genetic

value

into

an

enucleated

oocyte

from

a

different

animal

followed

by

electrical

activation

Subsequently

SCNT

requires

communication

of

enucleated

oocytes

with

the

donor

cell

nucleus

and

its

associated

centrosome

The

complex

molecular

regulation

and

functions

of

centrosomes

in

reproductive

cells

are

complicated

by

the

fact

that

somatic

cell

centrosomes

need

to

be

remodeled

by

the

enucleated

oocyte

-LRB-

reviewed

in

Sun

and

Schatten

-RRB-

which

is

crucially

important

as

the

donor

cell

's

centrosome

is

required

to

perform

all

functions

that

are

normally

fulfilled

by

the

blended

sperm

and

egg

centrosomal

material

Under

these

conditions

the

enucleated

oocyte

's

cytoplasmic

regulatory

machinery

is

required

to

regulate

the

donor

cell

centrosome

for

embryo-specific

functions

Centrosome

dysfunctions

may

occur

as

the

oocyte

's

cell

cycle

regulation

systems

are

different

from

those

used

by

somatic

cells

Indeed

multipolar

mitoses

resulting

from

supernumerary

centrosomes

have

been

reported

in

reconstructed

pig

oocytes

-LRB-

Zhong

et

al.

-RRB-

which

may

be

among

the

underlying

causes

for

decreased

cell

numbers

as

a

result

of

increased

mitotic

cell

death

While

blending

of

sperm

and

egg

centrosomal

material

into

a

functional

centrosome

after

fertilization

is

necessary

for

embryo-specific

functions

it

is

not

known

whether

the

donor

cell

centrosome

also

attracts

centrosomal

components

from

the

oocyte

or

whether

the

somatic

cell

centrosome

is

able

to

nucleate

and

organize

microtubules

for

oocyte-specific

functions

without

oocyte

centrosomal

components

Aberrant

composition

of

centrosome

proteins

will

result

in

abnormal

microtubule

organizations

as

has

been

discussed

for

cancer

cells

As

the

donor

cell

centrosome

contains

γ-tubulin

and

γ-TuRC

it

is

not

clear

whether

it

attracts

additional

γ-tubulin

from

the

oocyte

as

is

the

case

for

sperm

centrosomes

Analysis

of

cancer

cell

centrosomes

revealed

increased

γ-tubulin

associated

with

centrosomes

resulting

in

more

than

the

normal

number

of

microtubules

leading

to

aneuploidy

-LRB-

Lingle

et

al.

Schatten

et

al.

b

-RRB-

Increased

accumulation

of

microtubules

in

the

reconstructed

egg

may

also

play

a

role

in

centrosome

dysfunctions

resulting

in

abnormal

microtubule

organization

and

aneuploidy

-LRB-

Zhong

et

al.

Martin

et

al.

-RRB-

The

composition

of

the

centrosome

in

reconstructed

oocytes

is

not

known

and

needs

further

investigation

to

determine

whether

oocyte-stored

centrosome

proteins

are

recruited

to

the

donor

cell

centrosome

SCNT

also

challenges

nucleo-cytoplasmic

interactions

as

several

centrosome

proteins

such

as

NuMA

are

localized

in

the

nucleus

adding

further

complexities

to

centrosome

remodeling

The

oocyte

's

regulatory

systems

have

to

communicate

with

the

donor

cell

's

nuclear

and

centrosomal

material

as

many

of

the

cell-cycle-dependent

centrosome

proteins

are

of

nuclear

origin

and

depend

on

nucleo-cytoplasmic

interactions

for

cell

cycle-specific

and

developmentally

regulated

functions

Such

functions

include

governing

microtubule-mediated

translocation

of

mitochondria

for

spatio-temporal

requirements

for

ATP

translocation

of

macromolecular

complexes

vesicles

that

may

contain

enzymes

and

translocation

of

transient

centrosome-associated

proteins

that

are

needed

for

molecular

centrosome

restructuring

to

perform

cell

cycle-specific

centrosome

functions

We

do

not

yet

have

information

on

the

role

of

cyclin

B

in

centrosome

remodeling

in

reconstructed

embryos

which

will

be

important

information

to

obtain

as

cyclin

B

is

one

of

the

major

regulators

of

centrosomes

and

is

crucial

for

centrosome

maturation

and

for

cell

cycle-dependent

molecular

centrosome

dynamics

Cyclin

B

is

a

critical

regular

of

NuMA

association

with

centrosomes

and

its

release

from

spindle

poles

during

exit

from

mitosis

-LRB-

Saredi

et

al.

Merdes

and

Cleveland

Gehmlich

et

al.

-RRB-

In

contrast

to

mammalian

somatic

cells

in

which

cyclin

B

synthesis

increases

during

and

M

mainly

as

the

result

of

cyclin

B

gene

transcription

increase

in

embryonic

cells

cyclin

B

synthesis

is

constant

throughout

the

cell

cycle

and

cyclin

B

accumulation

is

the

result

of

decrease

in

its

degradation

rate

Taken

together

studies

of

centrosomes

in

reconstructed

embryos

will

provide

new

avenues

to

obtain

information

on

centrosomal

regulation

by

cytoplasmic

factors

Concluding

remarks

and

future

directions

Renewed

interest

in

centrosome

research

started

about

years

ago

when

it

was

shown

that

certain

autoimmune

sera

from

CREST

patients

would

reliably

label

centrosome

material

in

animal

cells

Since

then

rapid

progress

has

been

made

to

identify

specific

centrosome

proteins

and

determine

their

functions

which

led

to

renewed

appreciation

of

the

enormous

significance

that

centrosomes

carry

as

cellular

organelles

and

as

major

platform

for

cellular

regulation

Centrosome

dysfunctions

have

been

linked

to

various

human

genetic

diseases

-LRB-

reviewed

in

Badano

et

al.

-RRB-

and

centrosomes

can

directly

be

affected

by

environmental

and

genotoxic

stresses

Adverse

effects

on

centrosomes

will

result

in

multiple

cascades

of

cellular

dysfunctions

as

centrosomes

regulate

distribution

of

cellular

organelles

including

mitochondria

which

are

the

major

source

for

ATP

production

and

depend

on

microtubules

for

distribution

to

their

cell

cycle-specific

functional

destinations

Based

on

these

new

insights

it

is

easy

to

understand

that

centrosomes

are

linked

to

many

diseases

in

which

transport

along

microtubules

is

impaired

which

includes

Alzheimer

's

and

several

neurological

diseases

in

which

extensive

trafficking

along

microtubules

takes

place

This

area

of

research

with

a

new

focus

on

centrosomes

is

just

at

the

beginning

The

entire

field

of

aging

research

is

open

to

investigations

of

centrosomes

in

senescing

cells

Our

previous

studies

on

Drosophila

cells

in

culture

have

linked

multipolar

mitoses

to

cellular

senescence

-LRB-

Schatten

et

al.

-RRB-

and

further

studies

are

needed

to

explore

centrosome

regulation

in

aging

cells

Other

new

areas

of

research

include

the

effects

of

environmental

stresses

on

centrosomes

and

manipulation

and

exploitation

of

centrosomes

and

the

associated

microtubule

cytoskeleton

by

pathogens

which

has

been

explored

for

Toxoplasma

parasites

-LRB-

Coppens

et

al.

-RRB-

and

has

been

reviewed

by

Scaplehorn

and

Way

-LRB-

-RRB-

in

a

recent

book

dedicated

to

various

aspects

of

centrosome

research

-LRB-

Nigg

-RRB-

Taken

together

we

are

in

the

middle

but

also

at

the

beginning

of

new

appreciation

for

the

centrosome

as

key

organelle

and

as

main

station

for

directing

and

regulating

cellular

processes

through

its

microtubule

highway

system

and

further

research

will

undoubtedly

result

in

uncovering

and

perhaps

also

repairing

pathologies

related

to

centrosome

dysfunctions

centrosomes
microtubules
cell cycle
mitosis
centrosome proteins
centrosome functions
mtoc


Concerted action of Aurora B , Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function . Here we report that phosphorylation of H2A at threonine 119 -LRB- T119 -RRB- is enriched at centromere regions in Drosophila mitosis . We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres , while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis . Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset . Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore , multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation . Introduction In eukaryotes , genomic DNA is first packaged into nucleosomes and then organised into higher-order chromatin structures . Chromatin organisation is locally or globally changed in response to external and internal signals . The changes are required for executing critical biological functions , most notably in regulated gene expression and chromosome segregation . Various post-translational modifications take place on histones , mostly in their tail domains , and play critical roles in the regulation of chromatin structure and function , either directly or indirectly through the recruitment of specific chromatin binding proteins -LSB- 1 -RSB- . The importance of histone modifications in gene expression is well appreciated and has led to the hypothesis of ` the histone code ' , which proposes that the combination of various histone modifications defines the pattern of gene expression -LSB- 2 -RSB- . Upon entry into mitosis , chromatin undergoes dramatic morphological changes to form mitotic chromosomes . On mitotic chromosomes , centromeres form unique chromosomal domains which are crucial for chromosome segregation in two respects -LSB- 3 -RSB- . First , centromeres are sites which connect two sister chromatids through cohesins until anaphase . Second , they serve as the foundation for kinetochores which provide the sites for microtubule attachment . To execute these functions , centromeres need to adopt a specialised chromatin structure which also changes during the cell cycle , particularly at the entry into mitosis , at the metaphase -- anaphase transition and during exit from mitosis . Moreover , different regulation is also required for meiotic divisions to achieve a correct meiotic chromosome segregation pattern -LSB- 4 -RSB- . Recently a novel phosphorylation site was identified at threonine 119 -LRB- T119 -RRB- in the C-terminal tail of Drosophila H2A -LSB- 5 -RSB- . The site is conserved in H2A amongst eukaryotes -LRB- serine in yeasts -RRB- , but not in H2A variants , such as H2Av and H2AX . Here we show H2A T119 phosphorylation is enriched at centromeres during Drosophila mitosis . The Aurora B complex is required for this phosphorylation in centromeric regions , while Polo kinase suppresses phosphorylation by NHK-1 on chromosome arms . Inactivation of Cdc2 kinase is required for loss of centromeric phosphorylation at the metaphase-anaphase transition . Therefore , these mitotic kinases together control the temporal and spatial pattern of H2A phosphorylation at centromeres . Materials and methods Molecular and immunological techniques Standard immunological , DNA manipulation and protein techniques were followed throughout -LSB- 6,7 -RSB- . Mouse α-tubulin antibody DM1A -LRB- Sigma -RRB- was used as a loading control in western blots . For immunoblotting , peroxidase-conjugated secondary antibodies -LRB- Jackson Lab -RRB- were used and detected using an ECL kit -LRB- Amersham -RRB- . Primary antibodies used in this study include antibodies against Histone H2A -LRB- Upstate -RRB- , dH2A-pT119 -LSB- 5 -RSB- , phospho-H3 -LRB- Ser10 ; Upstate -RRB- , CID -LSB- 8 -RSB- , α-tubulin -LRB- DM1A ; Sigma -RRB- , GFP -LRB- 3E6 ; Molecular Probes -RRB- and Aurora B -LSB- 9 -RSB- . Immunofluorescence microscopy Culture and RNAi of S2 cells were carried out as described -LSB- 10,11 -RSB- . Effective depletion of target proteins was monitored by immunoblots or appearance of predicted phenotypes . S2 cells were immunostained as described with the exception that cells were fixed with 4 % paraformaldehyde in PBS for 5 min -LSB- 10 -RSB- . Larval central nervous systems were dissected from late third instar larvae and fixed with 11 % formaldehyde in 0.7 % NaCl as described -LSB- 12 -RSB- . Secondary antibodies conjugated with Cy3 or Alexa488 -LRB- Jackson Lab or Molecular Probes -RRB- were used at 1/250 -- 1/1000 dilution . S2 cells were transfected using Effectene Transfection Reagent -LRB- Qiagen -RRB- . Non-degradable cyclin B fused to GFP -LRB- pUASp-CBTPM-GFP -LSB- 13 -RSB- -RRB- was co-transfected with ubiquitin-GAL4 to induce expression . Transfected cells were identified by the presence of GFP . The presence of dH2A-pT119 on centromeres of segregated chromosomes -LRB- > 50 cells -RRB- was scored . Cultured cells were examined using a Plan-Apochromat objective lense -LRB- 100 × , 1.4 NA ; Zeiss -RRB- attached to an Axioplan2 -LRB- Zeiss -RRB- . Images were captured by a CCD camera -LRB- Orca ; Hamamatsu -RRB- using OpenLab2 -LRB- Improvision -RRB- . Larval central nervous systems were taken using a Plan-Apochromat lense -LRB- 63 × , 1.4 NA ; Zeiss -RRB- attached to an Axiovert 200 M -LRB- Zeiss -RRB- with a confocal scan head -LRB- LSM510meta ; Zeiss -RRB- . Confocal images were presented as a maximum intensity projection of the Z-stacks . All digital images were imported to Photoshop -LRB- Adobe -RRB- and adjusted for brightness and contrast . Phosphatase treatment For western blotting of phosphatase treated cell extract , cell extracts were obtained by resuspending S2 cells in lysis buffer -LRB- 150 mM NaCl , 20 mM Tris , 5 mM EDTA , 1 % NP-40 -RRB- with or without phosphatase inhibitors -LRB- 100 mM NaF , 2 μM okadaic acid , 100 mM β-glycero-phosphate , 15 mM p-nitrophenylphosphate -RRB- and incubating on ice for 10 min . Lambda phosphatase -LRB- NEB -RRB- was added to the cell extract without phosphatase inhibitors and both samples incubated for 30 min at 37 °C . 2 × SDS sample buffer was then added to the extracts and boiled for 3 min . Samples were then western blotted with anti-dH2A-pT119 to compare phospho-protein levels . In addition , cells immediately resuspended in 1 × SDS sample buffer were included for comparison . For phosphatase treatment of fixed cells for immunofluorescence with the anti-dH2A-pT119 antibody , cells were fixed with 4 % paraformaldehyde in PBS followed by incubation with lambda phosphatase for 1 h at 37 °C . Cells were then washed and immunostained as described above . Microscope images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment . Average pixel intensity of dH2A-pT119 staining on the DNA was measured in interphase and mitotic cells -LRB- 16 cells in 2 separate experiments -RRB- . Fly stocks Standard techniques for fly manipulation were followed -LSB- 14 -RSB- . All stocks were grown at 25 °C in standard cornmeal media . A null nhk-1 mutant -LRB- nhk-1E107 -RRB- used in this study was previously described -LSB- 15 -RSB- . Results H2A T119 phosphorylation is specific to centromeres in mitosis To examine the spatial and temporal control of H2A T119 phosphorylation in cells , we immunostained Drosophila S2 cells using an antibody which specifically recognises this phosphorylated form of H2A -LRB- anti-dH2A-pT119 -LSB- 5 -RSB- -RRB- . We found a dynamic change in the phosphorylation pattern of H2A during the cell cycle . In interphase , phosphorylation was present throughout the chromatin in the nucleus -LRB- Fig. 1A -RRB- . Interestingly , in mitosis , as the chromosomes begin to condense , phosphorylation was no longer spread throughout the chromatin but produced a more punctate pattern -LRB- Fig. 1B -RRB- . Co-staining with a centromeric marker CID -LRB- the CENP-A homologue ; -LSB- 8,16 -RSB- -RRB- revealed that in prometaphase and metaphase , phosphorylation was enriched in regions between and surrounding CENP-A positive regions , which we refer to as the centromeric regions -LRB- Figs. 1C -- E -RRB- . This phosphorylation became dramatically reduced at the onset of anaphase -LRB- Fig. 1F -RRB- . Phosphorylation only returned on decondensed chromatin at the end of mitosis . Specificity of the signal obtained by this phospho-H2A antibody was confirmed by treatment with lambda protein phosphatase . Lambda phosphatase treatment of S2 cell extracts eliminated a single band -LRB- which comigrates with H2A -RRB- recognised by the antibody on immunoblots -LRB- Supplementary Fig. 1 -RRB- . Furthermore , the immunofluorescent signals obtained by the phospho-H2A antibody were greatly reduced by lambda phosphatase treatment of fixed S2 cells -LRB- Supplementary Fig. 1 -RRB- . In syncytial embryos and oocytes , entire mitotic/meiotic chromosomes are stained with the anti-dH2ApT119 antibody -LSB- 5,17 -RSB- . To confirm that the phosphorylation pattern found in S2 cells is not specific to this cell line , we examined H2A phosphorylation in somatic cells of developing flies . The larval central nervous system -LRB- CNS -RRB- is the tissue most commonly used for the study of standard mitotic cell cycles , which have two gap phases and checkpoint regulation -LSB- 18 -RSB- . Immunostaining of larval CNSs revealed a similar temporal and spatial pattern of H2A T119 phosphorylation as found in S2 cells -LRB- Supplementary Fig. 2A -RRB- . Centromeric H2A T119 phosphorylation depends on Aurora B kinase Previously , the conserved protein kinase NHK-1 was identified as phosphorylating H2A T119 in vitro -LSB- 5 -RSB- . A female sterile mutation in NHK-1 greatly reduced phosphorylation at this site in oocytes , but not in follicle or nurse cells -LSB- 17 -RSB- . This indicated that NHK-1 is the major kinase responsible for this phosphorylation at least in the oocyte nucleus . To test whether NHK-1 is responsible for this phosphorylation in S2 cells , we examined whether depletion of this kinase by RNA interference -LRB- RNAi -RRB- affects the phosphorylation . Down-regulation of NHK-1 in S2 cells did not eliminate the signal of the phospho-H2A antibody in immunostaining -LRB- Supplementary Figs. 3 and 5 -RRB- . This result was further confirmed by immunostaining of larval CNSs from a null mutant of NHK-1 -LRB- nhk1E107 -LSB- 15 -RSB- -RRB- -LRB- Supplementary Fig. 2B -RRB- . These results indicated that either a residual amount of NHK-1 kinase is sufficient to phosphorylate this site or kinases other than NHK-1 can phosphorylate this site in the absence of NHK-1 . To identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation , we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation -LSB- 19 -RSB- . After Aurora B was depleted by RNAi , S2 cells were immunostained with phospho-H2A antibody . In Aurora B-depleted cells , the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms -LRB- Fig. 2A -RRB- . However , nuclear staining in interphase cells remained high -LRB- Supplementary Fig. 5 -RRB- , suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms . Aurora B kinase is part of at least two functionally distinct complexes -LSB- 19 -RSB- , a core complex -LRB- containing INCENP -RRB- and a larger complex -LRB- additionally containing Survivin and Borealin/DasraB -RRB- . To understand which complex is required for the H2A phosphorylation , we tested the requirement of other subunits for the phosphorylation . Depletion of any one of INCENP , Survivin and Borealin by RNAi greatly reduced H2A phosphorylation in centromeric regions in mitosis -LRB- Supplementary Fig. 4 -RRB- . Interphase phosphorylation was not affected in any of the cases . These results indicated that the large AuroraB complex is required for centromeric phosphorylation of H2A at T119 in mitosis . Polo kinase down-regulates the H2A phosphorylation on chromosome arms To further study the regulatory mechanism of the phosphorylation , we examined the role of the crucial mitotic regulator Polo kinase -LSB- 20 -RSB- . After Polo kinase was depleted by RNAi , S2 cells were immunostained with phospho-H2A antibody . Surprisingly , in Polo-depleted cells , H2A T119 phosphorylation was not restricted to centromeric regions in mitosis but remained at a high level on the entire chromosome arms -LRB- Fig. 2A -RRB- . Quantitative analysis indicated that the fluorescent signal from the phospho-H2A antibody on chromosome arms was dramatically increased in the absence of Polo kinase -LRB- Fig. 2B -RRB- . This result suggests that Polo kinase is directly or indirectly required for down-regulating H2A T119 phosphorylation on chromosome arms to enrich the phosphorylation at centromeric regions . Polo suppresses phosphorylation by the H2A kinase NHK-1 To identify the relationship between Aurora B and Polo actions , both of the kinases were depleted simultaneously . If a loss of Polo kinase misregulates Aurora B kinase , simultaneous depletion would suppress H2A T119 phosphorylation on chromosome arms . Immunostaining of cells depleted of both Aurora B and Polo showed a high level of phosphorylation on the entire chromosome arms -LRB- Fig. 2 -RRB- . This indicated that H2A T119 phosphorylation on chromosome arms induced by loss of Polo kinase was independent of Aurora B activity . Next we tested the relationship between Polo and the H2A kinase NHK-1 by co-depletion . We found that NHK-1 depletion suppresses H2A T119 phosphorylation on arms induced by a loss of Polo -LRB- Fig. 3 -RRB- . Quantitative analysis confirmed that the phospho-H2A signal on chromosome arms in Polo NHK-1 double depletions was decreased to a level comparable to that of the control or NHK-1 depletion . Finally we tested the phenotype of double depletion of Aurora B and NHK-1 . Like Aurora B single depletion , H2A T119 phosphorylation was greatly reduced from centromeric regions of mitotic chromosomes -LRB- Supplementary Fig. 5 -RRB- . These epistasis studies suggested that Polo functions upstream of NHK-1 to suppress H2A T119 phosphorylation , but is independent of Aurora B. Cyclin B degradation triggers a loss of H2A phosphorylation at initiation of anaphase Centromeric H2A T119 phosphorylation becomes greatly reduced at the onset of anaphase indicating a change in its regulation at this time . After alignment of all chromosomes , APC/Cdc20 triggers degradation of Cyclin B and securin , leading to inactivation of Cdc2 kinase and activation of separase which cleaves cohesin to initiate anaphase -LSB- 21 -RSB- . To separate Cyclin B degradation from securin degradation , we expressed non-degradable Cyclin B in S2 cells and examined H2A phosphorylation by immunostaining . As previously reported -LSB- 22,23,13 -RSB- , expression of non-degradable Cyclin B did not inhibit the onset of anaphase but prevented exit from mitosis , resulting in an accumulation of anaphase cells with overcondensed chromosomes . In cells expressing non-degradable Cyclin B , H2A phosphorylation was still retained at centromeric regions in most anaphase cells -LRB- Figs. 4A , B -RRB- . Therefore , we concluded that cyclin B degradation , not anaphase onset , is required for triggering loss of phosphorylation at the metaphase-anaphase transition . Discussion In this study , we found dynamic changes in H2A T119 phosphorylation during the Drosophila cell cycle . This phosphorylation is enriched at centromeric regions early in mitosis and lost at the onset of anaphase . In interphase , H2A T119 phosphorylation was found throughout chromatin . Furthermore , our evidence showed that the combined action of at least four conserved mitotic kinases is required for precise spatial and temporal regulation of H2A T119 phosphorylation -LRB- Fig. 4C -RRB- . Aurora B kinase is required for the enrichment of phosphorylation at centromeric regions in mitosis . Polo kinase is required for suppressing H2A phosphorylation by NHK-1 on chromosome arms . Furthermore , inactivation of Cdc2 kinase induced by Cyclin B degradation is required for the loss of centromeric phosphorylation at the onset of anaphase . Currently we do not know what the function of this H2A phosphorylation is in cells . In higher eukaryotes which have many copies of histone genes , the function of histone modifications has been studied only indirectly by down-regulating responsible modifying enzymes . Unfortunately this approach is not suitable for kinases as they are likely to have multiple substrates -LRB- for example , Cdc2 and Aurora B mediating H1 and H3 phosphorylation -RRB- . Centromeric distribution and regulation by conserved mitotic kinases may tempt us to speculate possible involvement of H2A T119 phosphorylation in chromosome segregation in mitosis . The phosphorylation might be important for generating or sensing tension between sister chromatids , or modes of microtubule attachment to kinetochores through the formation of centromere-specific chromatin or recruitment of centromere proteins during mitosis . A loss or misregulation of the H2A phosphorylation may be responsible for a subset of the highly pleiotropic phenotypes observed after down-regulation of Aurora B or Polo -LSB- 19,20 -RSB- . It would be a future challenge to define the precise roles of this H2A phosphorylation .

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