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TICB-1010; No. of Pages 10

Amorphous no more: subdiffractionview of the pericentriolar materialarchitectureVito Mennella1, David A. Agard1, Bo Huang2,3, and Laurence Pelletier4,5

1 Department of Biochemistry and Biophysics, HHMI and University of California, San Francisco, San Francisco, CA, USA2 Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA3 Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA4 Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada5 Department of Molecular Genetics, University of Toronto, Toronto, Canada

Review

The centrosome influences the shape, orientation andactivity of the microtubule cytoskeleton. The pericen-triolar material (PCM), determines this functionality byproviding a dynamic platform for nucleating microtu-bules and acts as a nexus for molecular signaling. Al-though great strides have been made in understandingPCM activity, its diffraction-limited size and amorphousappearance on electron microscopy (EM) have limitedanalysis of its high-order organization. Here, we outlinecurrent knowledge of PCM architecture and assembly,emphasizing recent super-resolution imaging studiesthat revealed the PCM has a layered structure made offibers and matrices conserved from flies to humans.Notably, these studies debunk the long-standing viewof an amorphous PCM and provide a paradigm to dissectthe supramolecular organization of organelles in cells.

Centrosome structure and functionThe microtubule cytoskeleton is a dynamic network offilaments comprising a/b-tubulin polymers that mediatesthe transport of organelles, protein complexes and segre-gation of genetic material through the mitotic spindleapparatus in eukaryotes. In most animal cells, the micro-tubule network is organized by the centrosome, a non-membrane-bound organelle comprising two molecular as-semblies with distinct but integrated functions: the cen-triole, a nine-fold symmetric cylindrical structureencircled by microtubule blades; and the PCM, tradition-ally described as an amorphous, electron-dense materialthat surrounds the centrioles [1,2]. Most cells have twocentrosomes, each containing a mother centriole and,following duplication, a daughter, whose dimensionscan reach approximately 200 nm in diameter and typicallybetween 200 and 400 nm in length depending on species,

0962-8924/$ – see front matter

� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.10.001

Corresponding author: Mennella, V. ([email protected]).Keywords: centrosomes; cilia; pericentriolar material; super-resolution microscopy;mitosis; cell cycle.

cell type, and the cell cycle phase. In cells with specializedfunctions, the size and number of centrioles can vary,reaching hundreds in number and microns in length [3].The two centrosomes, which differ in age as the centrioles,can be structurally distinguished by the presence of distaland subdistal appendages, which confer to the older of themother centrioles specialized microtubule-anchoring ca-pabilities [1,2,4,5]. In specialized non-dividing cells, themother centriole docks at the cell membrane and under-goes extensive structural modification to form the basalbody [6], which provides a template for the formation of themicrotubule-based axoneme, a structure critical for signaltransduction via cilia and for cellular movement as flagella[7].

The two structural elements of centrosomes, the cen-trioles and the PCM, have defined and interconnectedfunctional roles. The centrioles act as a structural scaffoldto promote the organization of the PCM [8–10]. Conversely,the primary functional role of the PCM is to anchor micro-tubules directly or through microtubule-nucleating centers(g-tubulin ring complexes [gTuRCs] [11]). At the onset ofmitosis, in a process termed centrosome maturation, thePCM increases in size and microtubule-nucleation capacitythrough an increase in the recruitment of gTuRCs from thecytosol. This leads to an increase in the nucleation of bothastral and spindle microtubules, which drive spindle for-mation, orientation, and, subsequently, cytokinesis [1].The PCM also plays a role in the duplication of centrioles[12,13] and probably in cilia formation and disassembly[14,15], further underscoring the interplay between thetwo assemblies. In addition to its more specific roles, thePCM broadly functions as a signaling and docking stationto regulate and redistribute protein assemblies throughmicrotubule transport by motor proteins or through asso-ciation with microtubule plus ends [16,17]. PCM proteinshave been implicated in cell cycle progression, proteindegradation, DNA damage, and hedgehog signaling [18–23], but it remains unclear to what extent the PCM scaffoldfunctions directly in the assembly and regulation of suchcomplexes.

Interestingly, the centrosome can be essential, critical,or dispensable for accurate division depending on the

Trends in Cell Biology xx (2013) 1–10 1

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Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x


specific cell and tissue and their reliance on oriented celldivisions. Thus eliminating the centrosome has severe butdistinct consequences for tissue organization, homeostasis,and development in different species. Mice with mutationsin many centrosomal proteins have dramatic developmen-tal defects, such as microcephaly. Conversely, flies andworms do not proceed beyond early embryogenesis withoutcentrosomes and die after a few cellular divisions [10,24–26]. Surprisingly, when flies survive the early embryonicstages with a parental contribution of centrosomes, theyare able to develop into adults and eventually die only aftereclosion due to lack of coordination [10]. Numerical andstructural centrosome aberrations are hallmarks of cellsfrom solid tumors and are thought to contribute to geneticinstability and cancer initiation and progression [2,26,27].Moreover, several hereditary diseases – collectively namedciliopathies – have been mapped to mutations in humangenes whose products encode centrosomal proteins with arole in cilia formation. This highlights the necessity tofurther comprehend how defects in centrosome organiza-tion and duplication are linked to disease states [5,26,28].

In recent years, mass spectrometry analyses of isolatedcentrosomes from different cells and organisms have de-fined the centrosome proteome [29,30]. A critical task incentrosome biology is to integrate this information intodefined protein molecular complexes and understand howtheir structure influences centrosomes activity in cells [31].Toward this end, studies by EM have provided insightsinto the ultrastructural organization of the centriole cart-wheel [32,33] and procentrioles [34,35] or gTuRCs [11].However, even with the use of electron tomography meth-ods, which early on revealed ring-shaped proteinaceousstructures embedded within the PCM [36], the moleculararchitecture and higher-order spatial organization of thePCM has remained, until recently, murky.

Here we describe the current understanding of centro-some architecture and the molecular mechanisms thatgovern PCM assembly. We emphasize recent efforts toexamine quantitatively the structure of the PCM usingsubdiffraction-resolution microscopy imaging methods –3D structured-illumination microscopy (3DSIM), stochas-tic optical-reconstruction microscopy (STORM) – and 3Dsubvolume alignment and averaging. Recently, four inde-pendent studies examined PCM spatial organization re-vealing the specific contribution and organization ofcentrosomal proteins critical for centrosome maturationin metazoans cells. Together they showed that the PCMcomprises two major domains with distinct molecularcomposition and architecture [37–40]. Notably, these stud-ies establish the higher-order organization of the PCM asan evolutionarily conserved property of centrosomes.

PCM architecture: historical perspective, roadblocks,and recent developmentsAlthough numerous studies have revealed importantstructural details of the centriole and basal bodies[32–34,41], our understanding of the architecture of thePCM has remained in its infancy for over a century. Thefirst description of the PCM dates back to the origin ofcentrosome biology in 1900, when T. Boveri defined it asthe unstructured region where microtubules appeared to

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originate [112]. Decades later, convincing evidence of thisinitial observation was provided by Gould and Borisy, whounequivocally showed that microtubule nucleation origi-nated from the PCM, thus defining its major functional role[42].

A central reason for this lack of progress can be attrib-uted to the rather homogeneous electron density of thePCM on EM, which precludes detailed studies of its orga-nization. Indeed, there is a striking paucity of evidencealluding to defined protein layers within the PCM. Mostnotable is the study of Paintrand and colleagues whereaveraging of electron micrographs exposed apparent fea-tures in the PCM region that were dependent on thecentrosome purification method [43]. EM studies on salt-stripped centrosomes of Drosophila and giant surf clamhave also revealed an insoluble protein matrix runningthroughout the PCM formed from 12–15 nm elements[44,45]. This structure is resistant to treatment with highconcentrations of chaotropic salts and is sufficient to allowfunctional reconstitution of gTuRC-mediated microtubulenucleation. Unfortunately, the molecular identity of thismatrix has remained elusive and the organization of itselements is unknown. This is a common issue in EMstudies, where imaging of intact centrosomes is relativelystraightforward, but labeling of specific proteins byimmuno-EM suffers from antigen disruption due to sec-tioning and antibody penetration issues.

Fluorescence microscopy studies based on deconvolu-tion microscopy have also begun challenging the notion ofan amorphous PCM organization. Unlike EM, fluorescencemicroscopy methods readily provide molecular specificitywith genetically encoded fluorescent tags and costainingwith multiple antibodies, while mostly preserving antigenstructure and accessibility. However, this method suffersfrom the very small size of the centrosome. Pioneeringwork from Doxsey and colleagues suggested that pericen-trin is organized in a lattice structure formed from multipleinterconnected rings [46] and Rattner and colleagues sug-gested the existence of a PCM tube around centriolescomprising the appendage proteins ninein and centriolin(CEP110) and pericentrin [47]. Yet, despite these efforts,our perception of the PCM as an amorphous, electron-dense structure has populated the textbooks and hasremained the dominant view until very recently.

Subdiffraction-resolution fluorescence microscopy haslately emerged as a powerful tool for the investigation ofthe architecture of protein complexes inside cells. Threedifferent technologies – 3DSIM, STORM and photoacti-vated localization microscopy (PALM), and stimulatedemission depletion (STED) – have been reliably employedto study diverse cellular structures by taking advantage ofdifferent principles to circumvent Abbe’s diffraction limit[48–51]. 3DSIM relies on modulation of the excitation lightby spatial patterning and, in its most practical implemen-tation, provides a resolution gain of only a factor of two(�125 nm in the x/y-plane and �250 nm in the z-plane, Box1), yet has the least stringent requirement for fluorophoreselection and readily accomodates multiple labeling [48].Conversely, PALM and STORM methods identify fluoro-phore position with higher resolution (�10 nm in the x/y-and �25 nm in the z-plane) by Gaussian fitting methods,

Centrioles

PCM fibers

PCM matrix

PCM architecture

Pericentrin B

Hu Pericentrin B378 KDa

1 3336

Coiled-coilKey:

744-909 3197-3336

CTD MD

NTD CTD

PACT

250nm

N2 N1

70-120 142-592

300 400 500 600 700

PCNT-N2

PCNT-N1

PCNT-MD

PCNT-CTD

mPCNT-GFP (BAC)

Diameter (nm)0

(A) (B)

(C)

PACT

Pericentrin/Plp

Cep152/Asl

NTD

NTD

CTD

Cep192/Spd-2 Cep215/Cnn

γTuRC

Key:Sas-6 CPAP Sas-4 mtsCentral hub Pericentrin Plp

Cep152 Asl Cep192 Spd-2 Cep215 CnnγTurc

CPAP/Sas-4

TRENDS in Cell Biology

Figure 1. Subdiffraction fluorescence microscopy view of the pericentriolar material (PCM) architecture. (A) Schematic representations of the architectural elements of the

interphase centrosome. The three layers of organization – centrioles and PCM fibers, and matrices – are represented separately to help visualization. The PCM is organized

in two major layers of proteins: (i) molecular fibers comprising the elongated coiled-coil proteins pericentrin/pericentrin-like protein (PLP) and Cep152/Asl have their C

termini near the centriole wall and their N termini extending toward the periphery; and (ii) a PCM matrix comprising Cep215/centrosomin (Cnn), g-tubulin, and Cep192/Spd-

2 molecules. See Figure 3 for a model with mitotic PCM. (B) Positional mapping of pericentrin domains on the interphase human centrosome. Top: Position of antibody/

probe positions on human pericentrin with predicted coiled-coil regions indicated. Bottom: Measurements of pericentrin diameter derived from 3D structured-illumination

microscopy (3DSIM) micrographs of human cells stained with anti-pericentrin domain-specific antibodies. The N terminus is positioned at a distance of >500 nm from the

center of the centriole. The PACT domain is position around �200 nm, a distance consistent with the centriole wall region, as measured from electron micrographs. (C) 2D

projections of averages of aligned 3D volumes of human centrosomes stained with antibodies against the C- and the N-terminus regions of pericentrin. Modified from

[37,38].



but necessitate more stringent control of fluorophore photo-activation and density [50,51]. Lastly, STED microscopy isbased on the concept of stimulated depleted emission, whichuses a powerful donut-shaped laser to bring fluorophoresaway from the excitation focal point thus reducing the effec-tive point-spread function (PSF) [49]. STED reaches similarresolution to PALM/STORM methods, but requires a moresophisticated optical setup and is more challenging in 3D. Allof these methods improve the resolution from two- to aboutten-fold compared with diffraction-limited fluorescence mi-croscopy thus allowing a quantum leap in the structuralanalysis of organelles and molecular complexes in cells,which are in large part beyond reach of classical structuralbiology methods (i.e., crystallography, cryo-EM) because oftheir size and need for cellular extraction and purification.

The analysis of the architecture of the PCM has been aparticularly challenging problem because of its biochemi-cal complexity (hundreds of components) including numer-ous large coiled-coil-containing proteins – most of whichare completely uncharacterized structurally – and itsdimensions at the boundary of the diffraction limit. Re-cently, several studies have started to exploit the resolvingpower of subdiffraction-resolution microscopy to examinecentrosome/basal body molecular assembly [52,113].

The mechanism of PCM assembly: scaffolding fibersand branching matrixIn novel systematic super-resolution microscopy studies,several groups demonstrated the existence of a PCM layerin the proximity of the centriole wall with a stunning,

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Box 1. Super-resolution fluorescence microscopy and 3D

volume data analysis

Super-resolution fluorescence-microscopy experiments generate

remarkable images, but, more importantly, they can provide a

substantial amount of quantitative positional information, all inside

the cell. The ensemble distribution of molecules and – in many

cases – domain localization and molecular orientation within

individual molecules can be obtained providing an unprecedented

snapshot of the organization of supramolecular complexes in cells

[37,38,87,88].

Recently, powerful image-processing and analysis tools inspired by

cryotomographic analyses have been implemented for the first time

in super-resolution microscopy. In essence, this method aligns

extracted 3D subvolumes in real space by translating, tilting, and in-

plane rotating the volumes to a reference by cross-correlation of voxel

intensities. The process is rendered unbiased by using an average of

the aligned volume as a reference for further rounds of refinement by

iterative alignment. The considerable advantage of this method is that

it provides excellent quantitative metrics of the spatial distribution of

centrosomal components with error in the range of a few nanometers,

rather than merely anecdotal examples [37]. This in turn favors

precise and unambiguous protein position assignment. The sub-

volume image stacks can be analyzed individually, to obtain

measurement variance and structural heterogeneity, as well as

through volume averages. Why the latter? The derivation of an

average structure enhances the structural similarities, while de-

emphasizing individual structural and/or staining heterogeneity. This

type of analysis leads to the discovery of shared structural features

and, via 3D clustering methods, could provide insights into distinct

structural states of the molecular assemblies [37].

Although 3D-alignment analysis substantially improves measure-

ment precision, the accuracy might be further improved by reducing

the distance between the antigen and the fluorophore. Toward this

end, an alternative to antibody primary–secondary complex (�15–

20 nm) is directly labeled anti-tag nanobodies, which are consider-

ably smaller probes (2–5 nm) and are available commercially [89].

An inherent limitation of all single-objective microscopic-imaging

methods is that the derived 3D volumes have anisotropic resolution.

That is, the resolution in the x- and y- planes is �2.5 times better than

that in the z-plane. This introduces a distortion in the observed

volumes and consequently reduces the accuracy of the distance

measurements. A simple way around this is to focus on those objects

oriented such that the measurements are in-plane (i.e., end-on view of

centrosomes) [38–40,89]. A more precise and general approach is to

correct for distortion of tilted volumes during the averaging by

properly merging the object information such that rotated objects

contribute axial resolution information largely missing in the other

views. This is best done by a hybrid real-space/Fourier-space

approach that can optimally make use of all information available

from each 3D subvolume in an unbiased manner [37].



highly ordered protein architecture in human and Dro-sophila cells [37–40]. The PCM proximal layer – named forits vicinity to the centriole wall – is readily visible oninterphase centrosomes and comprises several proteinsinvolved in centrosome maturation (Table 1). These com-ponents display distinctive distributions: some are orga-nized as apparent molecular fibers or toroids extendingaway from the centriole wall [pericentrin/pericentrin-likeprotein (PLP), Cep152/Asl], whereas others are organizedin a matrix [Cep192/Spd-2, Cep215/centrosomin (Cnn),and g-tubulin] (Figure 1).

A striking example of PCM molecular fibers is exempli-fied by pericentrin/PLP (Figure 1). Positional mappingusing probes against different domains followed by 3Dsubvolume alignment and averaging revealed toroids ofvarying diameters [37,38] (Figure 1B,C and Box 1). Target-ing the N-terminal region of pericentrin/PLP yielded larger

4

diameters than the C-terminal regions and probes target-ing the middle region produced intermediate results. Thissuggested that pericentrin/PLP adopts an extended con-formation and that the molecules are anchored at thecentriole wall region through their C-terminal PACT do-main, which had been shown previously to be sufficient forcentrosomal localization [53], whereas the N terminusprojects outward to the periphery. Pericentrin/PLP mole-cules extend a remarkable distance from the centriole wall(>150 nm in human cells; Figure 1B,C) [37–40]. Impor-tantly, STORM shows multiple PLP molecules formingclusters in the PCM region, which might follow the nine-fold symmetry of the centriole [37] (Figure 2A). Thesefindings suggest that pericentrin/PLP forms elongatedfibers in the PCM and support the notion that centriolesymmetry is not confined to its perimeter, but acts as anorganizing principle that extends into the PCM.

The coiled-coil protein Cep152/Asl has a similar orien-tation and distribution to pericentrin/PLP. Labeling thetwo termini shows that Cep152/Asl radiates outward withits C terminus at the centriole wall and the N terminusextending away [37,39]. Subdiffraction imaging shows thatPLP and Asl are mostly organized in an interleaved fash-ion with few areas of overlap, largely excluding the forma-tion of heteroligomeric structures through their coiled-coilregions [40] (Figure 2B).

Distinct from pericentrin/PLP and Cep152/Asl, mole-cules of Cep192/Spd-2 are distributed rather homoge-nously around the centriole wall; analysis with multipleantibodies against different regions of Cep192/Spd-2showed no clear polarity, suggesting that Cep192/Spd-2is a globular structure organized in a tightly packed ma-trix. This is consistent with the lack of extensive predictedcoiled-coil domains in its sequence [37–39]. Cep215/Cnnand g-tubulin molecules also appear randomly orientedaround the centriole wall and organized in a matrix thatcan extend to the outer region occupied by the N termini ofpericentrin/PLP [37–40]. Thus, in interphase centrosomestwo layers of organization of the PCM are present: aproximal layer of pericentrin/PLP and Cep152/Asl, whichform fibers extending up to hundreds of nanometers awayfrom the centriole wall; and, by contrast, a matrix ofinterdispersed Cep215/Cnn, g-tubulin, and Cep192/Spd-2molecules (Figure 1A).

In the early phases of mitosis, during centrosome matu-ration, the PCM proximal layer expands into a larger outermatrix to form the fully nucleating mitotic centrosome(Figure 3). Although nearly the same set of proteins ispresent, their architecture in the outermost PCM layerchanges. In particular, two components appear to adopt adifferent architecture. Pericentrin maintains its fiber-likeorganization around the mother centriole, but also expandsinto the PCM outer matrix in human cells to form the bulk ofthe PCM together with Cep215/Cnn [37–39]. Cep192/Spd-2instead goes from a tight toroidal organization around themother centriole to a matrix (Cep192)/reticular (Spd-2)structure in mitosis [37,38,40]. Notably, the PCM proximallayer is buried inside a large PCM matrix in humans andbecomes apparent only after PCM fragmentation – on, forexample, depletion of augmin activity – whereas it remainsvisible in Drosophila centrosomes, probably due to the

Table 1. Proteins implicated in centrosome maturation

Protein name Function in centrosome maturation Refs

TUB1G (Hu)

g-Tubulin (Dm, Ce)

Highly conserved protein required across species for the nucleation of microtubules. As part of

the gTuRC, forms a template for microtubule formation at the minus ends.

[11,90–93]

GCP71WD, Nedd1 (Hu)

Grip71WD (Dm)

Involved in targeting gTuRC to the spindle and the centrosome. It copurifies in a complex with

gTuRC, but also appears to have functions beyond that of a gTuRC-targeting factor.

[56,94–96]

Cep215,CDK5RAP2 (Hu)

Cnn (Dm)

Spd5 (Ce)

Essential protein for PCM maturation; it is the main candidate for a component of the PCM

matrix that provides direct binding to most of the gTuRCs in the PCM.

[60,97–99]

Cep192 (Hu)

Spd-2 (Dm)

Spd2 (Ce)

Key factor involved in both centriole duplication and PCM maturation in C. elegans, flies, and

human cells. It has two defined populations, one at the centriole wall and one in the PCM.

[57,64,65,100]

Pericentrin/kendrin (Hu)

PLP (Dm)

Large coiled-coil protein integral to the PCM involved in centrosome maturation across

metazoans. The C-terminal region contains the PACT domain, a conserved centrosome-

targeting region.

[14,62,101,102]

Cep152 (Hu)

Asterless (Dm)

Discovered in a mutant screen for male fertility in flies, it is primarily involved in centriole

duplication by defining the site of daughter centriole formation through Plk4 kinase interaction.

Asl might have a role in PCM stabilization/maintenance at the centriole.

[67,82–84,103,104]

Plk1 (Hu)

Polo kinase (Dm)

Conserved serine–threonine kinase involved in several aspects of cell division. It has been

shown to phoshorylate pericentrin directly to control centrosome maturation.

[56,58,105,106]

Aurora-A (Hu, Dm)

Air-1 (Ce)

Serine–threonine kinase implicated in several steps of cell division, including centrosome

maturation. Aurora-A is required for Cnn recruitment and phosphorylates TACC, which form a

complex with Msps to stabilize microtubules at the centrosome.

[70,76–78,107]

CPAP (Hu)

Sas-4 (Dm)

Conserved protein involved in centriole duplication and elongation. It might recruit PCM

proteins to the centriole during maturation as part of a large, multiprotein complex.

[9,80,108–110]

Cep97 (Hu)

Cep135 (Dm)

CP110 (Hu, Dm)

Rcd-5/6 (Dm)

Myb (Hu, Dm)

Calmodulin (Hu, Dm)

Map205 (Dm)

PP2A (Dm)

This list contains several other proteins implicated in centrosome maturation. Because their

functional role and conservation remain unclear at this stage they are not be discussed in this

review.

[59]



differences in PCM architecture compared with human cells(see below and [37,38,54,55]). 3D volume alignment andcross-correlation analysis (Box 1) of the components of theouter PCM matrix shows that they largely overlap, thussuggesting the formation of a matrix of interconnectingproteins within which specific domains of PCM componentsmight maintain their relative spatial positioning [37,38].

What is the functional interplay of the fibers and ma-trices of the proximal and outer layers of the PCM duringmaturation? Loss-of-function experiments suggest the ex-istence of separate but interdependent pathways for build-ing the PCM [56,57]. A scaffolding pathway, named afterpericentrin/PLP’s elongated structures, organizes thePCM proximal layer around the centriole wall and iscritical for proper 3D organization of the PCM outer layerduring maturation. A second pathway is mainly involved inmatrix formation and relies on Cep192/Spd-2, Cep215/Cnn, and g-tubulin, but does not appear to require peri-centrin/PLP [14,37,38,40,58,59,114].

In interphase centrioles, pericentrin/PLP appears to beat the top of the hierarchy of the PCM assembly of theproximal layer. Pericentrin/PLP is required to recruitCep215/Cnn [14,37,38,59,114], but likely not vice versa[38,60,61]. In turn, pericentrin is sufficient to recruitgTuRCs in the absence of Cep215 [38]. These activitiesmight be determined by pericentrin/PLP directly bindingto Cep215/Cnn and g-tubulin [46,62,63]. During centro-some maturation, pericentrin and Cep215/Cnn are the firstto expand into a large matrix in G2, but this is not sufficientfor full centrosome maturation, which requires Plk1

phosphorylation of pericentrin. Interestingly, only a sub-population of mitotic pericentrin located close to the cen-triole is phosphorylated and is required for recruitment ofCep192 and g-tubulin [58]. In addition, overexpression ofpericentrin during interphase is sufficient to recruitCep215 and build a massive PCM matrix similar to mitoticcentrosomes. These observations support the notion thatthe pericentrin layer proximal to the centriole wall has animportant role in PCM expansion under regulatory control.Conversely, in Drosophila, the pericentrin homolog PLPmaintains its fibrous/toroid structure around the mothercentriole, but does not expand into the outer PCM matrixduring centrosome maturation. This would suggest thatpericentrin might have acquired during evolution a poly-merizing molecular activity much like Cep215/Cnn thatDrosophila PLP lacks and explains the less dramatic effectof PLP depletion compared with pericentrin in centrosomematuration in flies [14,37,38,59,114].

Notably, a population of Cep215/Cnn, Cep192/Spd-2and g-tubulin is recruited to mitotic and interphasecentrosomes in the absence of pericentrin/PLP [37,38,56,57,59,61], indicating that Cep215/Cnn can self-assembleand/or have other molecular interactions independentlyfrom pericentrin (i.e., with Cep192/Spd-2) that are suffi-cient to form a reticular structure. This suggests the exis-tence of a second pathway that appears to be mainlyinvolved in matrix formation and relies on Cep192/Spd-2, Cep215/Cnn, and g-tubulin. Indeed, RNAi and geneticmutants show that Cep192/Spd-2 molecules are critical forCep215/Cnn matrix formation, thus making Cep192/Spd-2

5

200 nm

800 nm

100 nm

100 nm

Plp

(A)

(C)

(B)

Plk4

3D volume itera�ve alignmentby real space crosscorrela�on

Reference AverageAlignedUnaligned

Plp

Average radial distribu�on (nm)

Nor

mal

ized

pixe

l int

ensit

y

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 100 200 300 400

250 nm


Figure 2. Nanometer-scale organization of the pericentriolar material (PCM) proximal layer. (A) Stochastic optical-reconstruction microscopy (STORM) images of S2 cells

stained with antibodies against pericentrin-like protein (PLP), the Drosophila pericentrin ortholog. Left: End-on view. Right: Side view. Note the quasi-nine-fold symmetric

distribution of PLP. (B) 2D projections of 3D structured-illumination microscopy (3DSIM) images of Drosophila S2 cells stained with antibodies against Plk4 and PLP. Note

the interleaved distribution of PLP and Plk4 on interphase centrioles. The N termini of Asl have been shown to have a similar distribution to Plk4 on interphase S2 cells and

the two proteins interact directly in vitro. (C) Overview of the experimental scheme used for 3D volume alignment and averaging of subdiffraction-resolution images. See

Box 1. Modified from [37].



the main candidate to drive Cep215/Cnn oligomerization[57,64–66]. Surprisingly, in flies Spd-2 does not reach thefurther region of the PCM where Cnn forms a matrix, but itdoes have a reticular structure that might play a branchingrole in a subregion of the PCM [37,40]. Alternatively,Cep192/Spd-2 might promote PCM matrix formationthrough recruitment of a regulatory factor such as Plk1[40,66].

The existence of two separate pathways is further sup-ported by studies in humans and flies that show thatCep192/Spd-2 is important for Cep152/Asl recruitment[65,86], but not Plp [65]. In addition Plp and Asl are notrequired for the localization of each other [37,57,65,82].Altogether, these experiments suggest that pericentrin/PLP and Cep192/Spd-2 are indirectly linked in the PCMand provide more evidence for the notion that Cep192/Spd-2 and pericentrin/PLP lie in independent pathways forPCM organization through their mutual ability to interactwith Cep215/Cnn.

The experimental evidence summarized above is con-sistent with a combination of a template and self-organi-zation model for PCM formation [68]. The proximalinterphase PCM layer, which is formed from pericentrin/PLP fibers around the mother centriole, provides a molec-ular scaffold that recruits Cep215/Cnn. In turn, this scaf-fold provides a template for the mitotic outer matrixexpansion. This process is driven by more accumulationof Cep215/Cnn, Cep192/Spd-2, and, in human cells, peri-centrin.

6

The dynamics of PCM assembly and the role of kinasesHow is PCM recruitment regulated in space and timeduring centrosome maturation? As mentioned above, inhuman cells robust centrosome maturation requires Plk1-dependent phosphorylation of a subset of pericentrin mole-cules in the vicinity of the centriole wall and the subse-quent recruitment of Cep192 and g-tubulin [58].Consistently, Plk1/Polo is also localized in proximity tothe centriole wall [40,58]. Interestingly, Plk1/Polo is alsorequired for Cnn phosphorylation, suggestive of a directrole for these kinases in Cnn regulation during PCMexpansion [40,56,58,59]. Fluorescence recovery afterphotobleaching (FRAP) experiments in fly embryos showthat Cnn molecules are dynamically associated with thecentrosome and are first recruited near the centriole,before spreading to the centrosome periphery [69]. Bycontrast, FRAP studies in fly embryos indicate that thecentriole-associated PACT domain of centriolar PLP doesnot exchange rapidly [14]. Because the pericentrin/PLPpopulation at the centriole wall is important for matura-tion, it would be interesting to examine whether PLP canalso be phosphorylated by Polo (like its mammalian coun-terpart), if so possibly unveiling a conserved regulationmechanism in PCM recruitment, in which phosphorylatedpericentrin/PLP at the centriole wall provides structuralscaffolding for PCM expansion. Interestingly, PLP loss offunction has opposite effects on PCM recruitment on thedaughter (apical) compared to the mother (basal) inter-phase centrosomes in Drosophila neuroblasts suggesting a

Centrosomematura�on

G2/M

Cep215/CnnCep192/Spd-2

pericentrinγTurc

Pericentrin/Plp

NTD

PACT

Centrosome matura�on

Interphase PCM

Mature PCM


Figure 3. Pericentriolar material (PCM) architecture during centrosome

maturation. Schematic representation of a mammalian centrosome during

centrosome maturation. Note the expansion of the PCM proximal layer of

interphase cells during G2/M through the formation of an outer matrix of

Cep215, pericentrin, and Cep192 molecules. g-Tubulin ring complexes (gTuRCs)

are embedded within the PCM and promote microtubule nucleation during

mitosis. By contrast, PCM expansion in Drosophila does not require the formation

of a matrix comprised of the pericentrin homolog PLP, but only of its centriole

associated component.



special form of regulation relying on Polo localization instem cells whose mechanism remains unclear [112].

The serine–threonine kinase Aurora-A is also implicat-ed in centrosome maturation [70–72]. Although its activityis required for mitotic entry through regulation of Cdk1and Plk1 kinases in a feedback loop through the proteinBora [73–75], its function in centrosome maturationappears distinct from Plk-1. Aurora-A and Cnn are mutu-ally dependent for their centrosomal recruitment duringmaturation and this activity is mediated through a directinteraction between Aurora-A and a Cnn C-terminal re-gion [76]. Aurora-A also stabilizes microtubule growth byphosphorylation of TACC, a protein in complex at thecentrosome with the microtubule regulator MSPS[77,78]. It will be of interest to better understand theinterplay between these kinases and the full set of sub-strates they regulate during centrosome maturation.

Recent work suggests that some PCM components im-plicated in centrosome maturation (PLP, Asl, Cnn) are partof a large protein complex containing tubulin and Sas-4,which acts as an attachment factor to the centriole wall[79,80]. Although these complexes were shown to associatein embryonic extracts, it remains unclear whether they areassembled in the cytoplasm before centrosomal loading orwhether they derive from PCM disruption during purifica-tion. Although PLP, Asl, and Cnn are present together onmother centrioles in Drosophila interphase cells, these

PCM proteins are recruited to daughters at distinct timesduring mitosis, with Sas-4 recruited first to the daughtercentriole and subsequently, but not simultaneously, Asland PLP around metaphase, followed by the outer matrixcomponents Cnn and g-tubulin [37]. It is possible thatDrosophila cells and embryos have different timing mech-anisms for recruitment of these proteins, owing to thedifferent time scales of cell division. Alternatively, it ispossible that only a small subpopulation of these proteinsmight be forming a bona fide complex while transportedtogether to the centrosome or when all are located at thecentrosome after docking [16,81].

Platforms and gates in centriole duplicationStudies from worms to humans suggest that mother cen-trioles act as an assembly ‘platform’ for the formation ofdaughter centrioles during their duplication. This processcomprises the sequential recruitment of Cep192/Spd-2,Cep152/Asl, Plk4, Sas-6, and CPAP/Sas-4, which allowsprocentriole assembly [34,82–85]. Subdiffraction-resolu-tion analysis together with 3D subvolume alignment andaveraging of centrosomes in G1 and G2 shows that Cep152/Asl and pericentrin/PLP undergo a pronounced change inarchitecture by forming a zone of exclusion on mothercentrioles, which correlates with the position where thedaughter emerges [37,38]. These observations suggest amultistep mechanism that involves changes in PCM mo-lecular fiber organization. This includes the selection of thesite on the mother centriole where daughter centrioles willemerge on the recruitment of Cep152/Asl structures byPlk4 and subsequent disassembly of a subpopulation ofCep152/Asl and pericentrin/PLP, to allow growth of thedaughter centriole [37–39,86]. This molecular gap, which ispresent in human and flies, is observed in Asl and PLPstructures when labeled using anti-gfp nanobodies (�2 nmin size), thus probably excluding the possibility of lack ofantigen accessibility to antibody binding (unpublishedobservations).

Concluding remarksOur understanding of PCM architecture has changed sub-stantially through the recent advancements of subdiffrac-tion imaging. Although the existence of an organized layerof proteins around centrioles (PCM tube) [4,15,47] hadbeen previously hypothesized, the findings highlightedin this review reveal the existence of multiple layers inthe PCM. More precisely, they expanded the view of peri-centrin architecture – previously described as a lattice-likestructure [46] – and function by revealing the dual natureof pericentrin distribution (mitotic matrix-like versus in-terphase fibers) as well as the orientation of its moleculeswithin the PCM. In addition, they provide a systematicanalysis – with a quantitative method with nanometerprecision – to establish the position and orientation of alarge number of proteins critical for various facets ofcentrosome biogenesis. These studies further explain thestructural heterogeneity of the components closely associ-ated with the centriole wall during the cell cycle by show-ing that their state (open versus closed) is correlated withdaughter centriole formation. Lastly, and critically, theyprovide a conceptual framework that will greatly facilitate

7

Box 2. Outstanding questions

� Several fundamentally important but unanswered questions re-

main. Is Cep215/Cnn capable of self-assembly? What are the precise

molecular roles of the other PCM components like pericentrin/PLP

and Cep192/Spd-2 in building the PCM matrix around interphase

centrioles, how are these relationships modulated during centro-

some maturation, and how are they regulated by the cell cycle

machinery? Are Cep192/Spd-2 and pericentrin/PLP required to alter

the connectivity, the orientation, and/or the kinetics of Cep215/Cnn

assembly? To answer these questions it will be important to

determine whether the set of molecular interactions underpinning

PCM organization during interphase differs from that in mitosis.

Although pericentrin/PLP and Cep215/Cnn interactions appear to be

maintained throughout the cell cycle, evidence suggests that Spd-2

might play a major role in PCM assembly during mitosis but not

interphase [38]. Recent work suggests that Cep192 participates in

the phospho-regulation of NEDD1 and controls its ability to

associate with and recruit g-tubulin to mitotic centrosomes, raising

the tantalizing possibility that Cep192 contributes to PCM activation

at the onset of mitosis [111]. A systematic biochemical analysis of

the interactions with recombinant material or subcomplexes

purified from cell extracts at various stages of centrosome

assembly will be necessary to answer these questions directly.

Considering the importance of microtubule organization and

anchoring in specialized cellular processes, including stem-cell

division, it would be important to analyze the similarities and

differences in PCM organization during interphase in cells about to

undergo symmetric versus asymmetric divisions [5,114].

� During metazoan evolution, pericentrin/PLP has acquired the ability

to be recruited not only at the centriole wall as a fiber (in human

and Drosophila cells) but also as a matrix component in the outer

PCM during maturation (in human cells). This more recently

acquired property of pericentrin is under regulatory control. Indeed,

overexpression of pericentrin in human cells can lead to matrix

formation in interphase centrosomes, which in normal conditions

have only a fiber/toroid distribution. Interestingly, this function is

not required to make the outer matrix in Drosophila – or in

Caenorhabditis elegans, which seems to lack a pericentrin homolog

altogether. This would suggest that the original primary role of

pericentrin/PLP is to provide an interphase scaffold for PCM

recruitment and that later in evolution it gained additional

functionality to facilitate PCM expansion in the outer matrix.

� Pericentrin/PLP is expressed in metazoans in many isoforms and

splicing variants [14,102]. Super-resolution imaging seems unable

to resolve the individual isoforms, suggesting that the different

pericentrin/PLP species are arranged in close proximity or bundle

together into a multimolecular fiber at the centrosome with a

symmetry likely dictated by the centriole nine-fold structure (see

Figure 2D in main text) [37]. In vitro biochemical and structural

analysis will be critical to demonstrate the existence of a

pericentrin/PLP multimeric structure and to define the biochemical

determinants of its formation and the role of the different isoforms

in higher-order PCM matrix organization.

� Cep152/Asl on interphase centrosomes is located in the same PCM

domain as pericentrin/PLP and has a similar elongated orientation,

but neither pericentrin/PLP nor Cep152/Asl is required for the

recruitment of the other, implying independent anchoring to the

centriole wall [37,86]. Do the Cep152/Asl elongated structures have

a role in organizing the PCM or only in providing a platform for

duplication? Mutant analysis of Drosophila spermatocytes suggests

that Asl is indeed necessary for PCM stabilization [67,104], whereas

RNAi studies suggest that it is not [40]. One possibility is that Asl

has an indirect role in PCM organization by recruiting Spd-2 at the

centriole wall, which might affect Spd-2 matrix organization.

Further studies are required to address this issue.

� How do pericentrin/PLP and Cep152/Asl fibers bind to the centriole

wall? Although Asl has been shown to bind Sas-4 directly [82], it

remains unclear how PLP is anchored to the centriole wall. Because

pericentrin/PLP appears to be distributed symmetrically around the

centriole, it is likely to take advantage of the microtubule doublets/

triplets, directly or through an attachment factor, or of the space

between [37,38]. Interestingly, pull-down interaction experiments

suggested that Sas-4 N termini might link PLP to the centriole wall [80].

� Recent discoveries defined Cep215 as the major interaction partner

and regulator of gTuRC activity in the PCM [60,99]. In addition, the

N-terminal domain (NTD) of pericentrin/PLP has been shown to

bind gTuRC directly, albeit weakly, through GCP2/3 [46,63], where it

may help activate gTuRCs for microtubule nucleation. This putative

globular domain does not seem to be necessary for PCM

maturation and its precise role remains unclear [37]. Further studies

will be required to assess whether pericentrin and gTuRC are

indeed part of a bona fide complex and, if so, what their functional

role is in centrosome maturation and PCM organization.



understanding of centrosome maturation at the structurallevel. The future challenge is to integrate the composi-tional, functional, and structural data into a coherent viewof PCM assembly – see Box 2 for analysis of outstandingquestions. A major task is to bridge the nanometer-resolu-tion information obtained from super-resolution microsco-py with the Angstrom resolution maps derived fromclassical structural methods. Integral to this task is thegeneration of probes targeting various regions of all cen-trosomal proteins and the isolation or reconstitution ofprotein complexes from purified components or extracts.In addition, the molecular role of individual components inthe kinetics of PCM assembly will be clearly defined via invitro reconstitution, enabling the basic principles of PCMformation to be elucidated. These studies should be guidedby parallel efforts in cells aimed at defining a high-resolu-tion map of the dynamics of PCM proteins and a compre-hensive analysis of their regulatory networks.

In conclusion, recent studies highlighted here demon-strate the power of combining multimodal subdiffraction-resolution microscopy with quantitative image analysis toreveal molecular-scale details of organelle architecture.Importantly, they defined a fundamental map of PCM

8

organization and revealed a functional framework to dis-sect how its organization is achieved. In-depth understand-ing of the centrosome and basal body architecture andfunction is crucial for the discovery and validation ofprotein targets as determinants of disease.

AcknowledgmentsThe authors sincerely apologize to authors whose studies they could notcite due to space constraints.

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amorphous no more: subdiffraction view of the pericentriolar … · amorphous no more:...

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