Microtubule organization in three-dimensional confined geometries: evaluating the role of elasticity through a combined in vitro and modeling approach

Microtubules or microtubule bundles in cells often grow longer than the size of the cell, which causes their shape and organization to adapt to constraints imposed by the cell geometry. We test the reciprocal role of elasticity and confinement in the organization of growing microtubules in a confining box-like geometry, in the absence of other (active) microtubule organizing processes. This is inspired, for example, by the cortical microtubule array of elongating plant cells, where microtubules are typically organized in an aligned array transverse to the cell elongation axis. The method we adopt is a combination of analytical calculations, in which the polymers are modeled as inextensible filaments with bending elasticity confined to a two-dimensional surface that defines the limits of a three-dimensional space, and in vitro experiments, in which microtubules are polymerized from nucleation seeds in microfabricated chambers. We show that these features are sufficient to organize the polymers in aligned, coiling configurations as for example observed in plant cells. Though elasticity can account for the regularity of these arrays, it cannot account for a transverse orientation of microtubules to the cell's long axis. We therefore conclude that an additional active, force-generating process is necessary to create a coiling configuration perpendicular to the long axis of the cell.     

Microtubule cytoskeleton and morphogenesis in the amoebae of the myxomycete Physarum polycephalum

Summary— The amoebae of the myxomycete Physarum polycephalum are of interest in order to analyze the morphogenesis of the microtubule and microfilament cytoskeleton during cell cycle and flagellation. The amoebal interphase microtubule cytoskeleton consists of 2 distinct levels of organization, which correspond to different physiological roles. The first level is composed of the 2 kinetosomes or centrioles and their associated structures. The anterior and posterior kinetosomes forming the anterior and posterior flagella are morphologically distinguishable. Each centriole plays a role in the morphogenesis of its associated satellites and specific microtubule arrays. The 2 distinct centrioles correspond to the 2 successive maturation stages of the pro-centrioles which are built during prophase. The second level of organization consists of a prominent microtubule organizing center (mtoc 1) to which the anterior centriole is attached at least during interphase. This mtoc plays a role in the formation of the mitotic pole. These observations based on ultrastructural and physiological analyses of the amoebal cystoskeleton are now being extended to the biochemical level. The complex formed by the 2 centrioles and the mtoc 1 has been purified without modifying the microtubule-nucleating activity of the mtoc 1. Several microtubule-associated proteins have been characterized by their ability to bind taxol-stabilized microtubules. Their functions (e.g., microtubule assembly, protection of microtubules against dilution or cold treatment, phosphorylating and ATPase activities) are under investigation. These biochemical approaches could allow in vitro analysis of the morphogenesis of the amoebal microtubule cytoskeleton.     

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.     

Ordering microtubules

How do cells order their cytoplasm? While microtubule organizing centers have long been considered essential to conferring order by virtue of their microtubule nucleating activity, attention has currently refocused on the role that microtubule motors play in organizing microtubules. An intriguing set of recent findings⁽¹⁾ reveals that cell fragments, lacking microtubule organizing centers, rapidly organize microtubules into a radial array during organelle transport driven by the microtubule motor, cytoplasmic dynein. Further, interaction of radial microtubules with the cell surface centers the array, revealing that centering information resides not with centrosomes but with organized microtubules.     

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.     

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.     

Microtubule cortical array organization and plant cell morphogenesis

Plant cell cortical microtubule arrays attain a high degree of order without the benefit of an organizing center such as a centrosome. New assays for molecular behaviors in living cells and gene discovery are yielding insight into the mechanisms by which acentrosomal microtubule arrays are created and organized, and how microtubule organization functions to modify cell form by regulating cellulose deposition. Surprising and potentially important behaviors of cortical microtubules include nucleation from the walls of established microtubules, and treadmilling-driven motility leading to polymer interaction, reorientation, and microtubule bundling. These behaviors suggest activities that can act to increase or decrease the local level of order in the array. The SPIRAL1 (SPR1) and SPR2 microtubule-localized proteins and the radial swollen 6 (rsw-6) locus are examples of new molecules and genes that affect both microtubule array organization and cell growth pattern. Functional tagging of cellulose synthase has now allowed the dynamic relationship between cortical microtubules and the cell-wall-synthesizing machinery to be visualized, providing direct evidence that cortical microtubules can organize cellulose synthase complexes and guide their movement through the plasma membrane as they create the cell wall.     

The Centrosomal Linker and Microtubules Provide Dual Levels of Spatial Coordination of Centrosomes

The centrosome is the principal microtubule organizing center in most animal cells. It consists of a pair of centrioles surrounded by pericentriolar material. The centrosome, like DNA, duplicates exactly once per cell cycle. During interphase duplicated centrosomes remain closely linked by a proteinaceous linker. This centrosomal linker is composed of rootletin filaments that are anchored to the centrioles via the protein C-Nap1. At the onset of mitosis the linker is dissolved by Nek2A kinase to support the formation of the bipolar mitotic spindle. The importance of the centrosomal linker for cell function during interphase awaits characterization. Here we assessed the phenotype of human RPE1 C-Nap1 knockout (KO) cells. The absence of the linker led to a modest increase in the average centrosome separation from 1 to 2.5 μm. This small impact on the degree of separation is indicative of a second level of spatial organization of centrosomes. Microtubule depolymerisation or stabilization in C-Nap1 KO cells dramatically increased the inter-centrosomal separation (> 8 μm). Thus, microtubules position centrosomes relatively close to one another in the absence of linker function. C-Nap1 KO cells had a Golgi organization defect with a two-fold expansion of the area occupied by the Golgi. When the centrosomes of C-Nap1 KO cells showed considerable separation, two spatially distinct Golgi stacks could be observed. Furthermore, migration of C-Nap1 KO cells was slower than their wild type RPE1 counterparts. These data show that the spatial organization of centrosomes is modulated by a combination of centrosomal cohesion and microtubule forces. Furthermore a modest increase in centrosome separation has major impact on Golgi organization and cell migration.     

Cell edges accumulate gamma tubulin complex components and nucleate microtubules following cytokinesis in Arabidopsis thaliana

Microtubules emanate from distinct organizing centers in fungal and animal cells. In plant cells, by contrast, microtubules initiate from dispersed sites in the cell cortex, where they then self-organize into parallel arrays. Previous ultrastructural evidence suggested that cell edges participate in microtubule nucleation but so far there has been no direct evidence for this. Here we use live imaging to show that components of the gamma tubulin nucleation complex (GCP2 and GCP3) localize at distinct sites along the outer periclinal edge of newly formed crosswalls, and that microtubules grow predominantly away from these edges. These data confirm a role for cell edges in microtubule nucleation, and suggest that an asymmetric distribution of microtubule nucleation factors contributes to cortical microtubule organization in plants, in a manner more similar to other kingdoms than previously thought.     

Ste20-related protein kinase LOSK (SLK) controls microtubule radial array in interphase

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

Extending the Microtubule/Microfibril Paradigm : Cellulose Synthesis Is Required for Normal Cortical Microtubule Alignment in Elongating Cells

The cortical microtubule array provides spatial information to the cellulose-synthesizing machinery within the plasma membrane of elongating cells. Until now data indicated that information is transferred from organized cortical microtubules to the cellulose-synthesizing complex, which results in the deposition of ordered cellulosic walls. How cortical microtubules become aligned is unclear. The literature indicates that biophysical forces, transmitted by the organized cellulose component of the cell wall, provide a spatial cue to orient cortical microtubules. This hypothesis was tested on tobacco (Nicotiana tabacum L.) protoplasts and suspension-cultured cells treated with the cellulose synthesis inhibitor isoxaben. Isoxaben (0.25–2.5 μm) inhibited the synthesis of cellulose microfibrils (detected by staining with 1 μg mL⁻¹ fluorescent dye and polarized birefringence), the cells failed to elongate, and the cortical microtubules failed to become organized. The affects of isoxaben were reversible, and after its removal microtubules reorganized and cells elongated. Isoxaben did not depolymerize microtubules in vivo or inhibit the polymerization of tubulin in vitro. These data are consistent with the hypothesis that cellulose microfibrils, and hence cell elongation, are involved in providing spatial cues for cortical microtubule organization. These results compel us to extend the microtubule/microfibril paradigm to include the bidirectional flow of information.     

Inhibition of Cell Migration and Cell Division Correlates with Distinct Effects of Microtubule Inhibiting Drugs

Drugs that target microtubules are thought to inhibit cell division and cell migration by suppressing dynamic instability, a “search and capture” behavior that allows microtubules to probe their environment. Here, we report that subtoxic drug concentrations are sufficient to inhibit plus-end microtubule dynamic instability and cell migration without affecting cell division or microtubule assembly. The higher drug concentrations needed to inhibit cell division act through a novel mechanism that generates microtubule fragments by stimulating microtubule minus-end detachment from their organizing centers. The frequency of microtubule detachment in untreated cells increases at prophase suggesting that it is a regulated cellular process important for spindle assembly and function. We conclude that drugs produce differential dose-dependent effects at microtubule plus and minus-ends to inhibit different microtubule-mediated functions.     

Cell Cycle Regulation of Microtubule Interactomes: Multi-layered Regulation Is Critical for the Interphase/Mitosis Transition

Microtubules dramatically change their dynamics and organization at the entry into mitosis. Although this change is mediated by microtubule-associated proteins (MAPs), how MAPs themselves are regulated is not well understood. Here we used an integrated multi-level approach to establish the framework and biological significance of MAP regulation critical for the interphase/mitosis transition. Firstly, we applied quantitative proteomics to determine global cell cycle changes in the profiles of MAPs in human and Drosophila cells. This uncovered a wide range of cell cycle regulations of MAPs previously unidentified. Secondly, systematic studies of human kinesins highlighted an overlooked aspect of kinesins: most mitotic kinesins suppress their affinity to microtubules or reduce their protein levels in interphase in combination with nuclear localization. Thirdly, in-depth analysis of a novel Drosophila MAP (Mink) revealed that the suppression of the microtubule affinity of this mitotic MAP in combination with nuclear localization is essential for microtubule organization in interphase, and phosphorylation of Mink is needed for kinetochore-microtubule attachment in mitosis. Thus, this first comprehensive analysis of MAP regulation for the interphase/mitosis transition advances our understanding of kinesin biology and reveals the prevalence and importance of multi-layered MAP regulation.Microtubules are universally found in eukaryotic cells and are involved in diverse processes including cell division, polarity, and intracellular transport. A striking feature of microtubules is that they change their dynamics and organization depending on cellular contexts. Proteins that interact with microtubules, collectively called microtubule-associated proteins (MAPs),¹ are considered to play a major role in determining microtubule dynamics and organization.Although MAPs in general lack recognizable sequence motifs, many MAPs from various sources have been successfully identified by means of biochemical purification followed by mass spectrometry (1–4). However, functional analysis is more problematic, as hundreds of MAPs can interact with microtubules. In addition, multiple MAPs have functional redundancy (5–7), making their biological function often difficult to determine, which results in their importance being grossly underappreciated. Furthermore, it is challenging to understand how MAPs collectively determine the diverse organization and dynamics of microtubules in different cells.One of the most dramatic changes of microtubule organization is found at the transition from interphase to mitosis. During mitosis, microtubules are much more dynamic and are organized into a dense bipolar structure, the spindle, whereas microtubules in interphase are less dynamic and are arranged in a radial array. This transition is rapid and is thought to reflect mainly a change in the activities of both motor and nonmotor MAPs (8); however, we do not have sufficient knowledge of how MAPs themselves are regulated. It is crucial to identify and understand the regulation of MAPs whose activities change in the cell cycle, and how they collectively change microtubule dynamics and organization. Misregulation of such MAPs could interfere with chromosome segregation or cell polarity and potentially contribute to oncogenesis (9). Also, this misregulation can be used to elucidate important functions that are masked due to functional redundancy.We hypothesize that some proteins bind to microtubules only during mitosis and are released from microtubules in interphase. The binding of such proteins to spindle microtubules in mitosis could collectively trigger the formation of the functional spindle, and, of equal importance, removing such proteins from microtubules at the mitotic exit could be essential for disassembling the spindle and proper organization and/or function of interphase microtubules. Conversely, some proteins may bind to microtubules specifically during interphase. No studies have been reported that systematically identify proteins whose microtubule-binding activities change between interphase and mitosis.Here we report a combined approach integrating three levels of analyses to gain insights into how MAPs are regulated as a whole to drive microtubule reorganization at the transition between interphase and mitosis. Firstly, we applied proteomics to determine the quantitative change of the global MAP profile between mitosis and interphase in both human and Drosophila cells. Secondly, we systematically analyzed the human kinesin superfamily for cell cycle localization in relation to microtubule association to gain insight into the general principle of MAP regulation in the cell cycle. Thirdly, we focused on one novel Drosophila MAP to understand the molecular mechanism and biological significance of MAP regulation. This integrated approach has provided the framework of MAP regulation critical for the interphase/mitosis transition.     

Fluctuation Analysis of Centrosomes Reveals a Cortical Function of Kinesin-1

The actin and microtubule networks form the dynamic cytoskeleton. Network dynamics is driven by molecular motors applying force onto the networks and the interactions between the networks. Here we assay the dynamics of centrosomes in the scale of seconds as a proxy for the movement of microtubule asters. With this assay we want to detect the role of specific motors and of network interaction. During interphase of syncytial embryos of Drosophila, cortical actin and the microtubule network depend on each other. Centrosomes induce cortical actin to form caps, whereas F-actin anchors microtubules to the cortex. In addition, lateral interactions between microtubule asters are assumed to be important for regular spatial organization of the syncytial embryo. The functional interaction between the microtubule asters and cortical actin has been largely analyzed in a static manner, so far. We recorded the movement of centrosomes at 1 Hz and analyzed their fluctuations for two processes—pair separation and individual movement. We found that F-actin is required for directional movements during initial centrosome pair separation, because separation proceeds in a diffusive manner in latrunculin-injected embryos. For assaying individual movement, we established a fluctuation parameter as the deviation from temporally and spatially slowly varying drift movements. By analysis of mutant and drug-injected embryos, we found that the fluctuations were suppressed by both cortical actin and microtubules. Surprisingly, the microtubule motor Kinesin-1 also suppressed fluctuations to a similar degree as F-actin. Kinesin-1 may mediate linkage of the microtubule (+)-ends to the actin cortex. Consistent with this model is our finding that Kinesin-1-GFP accumulates at the cortical actin caps.     

Identification of a kinesin-like microtubule-based motor protein in Dictyostelium discoideum.

Dictyostelium discoideum, a unicellular eukaryote amenable to both biochemical and genetic dissection, provides an attractive system for studying microtubule-based transport. In this work, we have identified microtubule-based motor activities in Dictyostelium cell extracts and have partially purified a protein that induces microtubule translocation along glass surfaces. This protein, which sediments at approximately 9S in sucrose density gradients and is composed of a 105 kd polypeptide, generates anterograde movement along microtubules that is insensitive to 5 mM NEM (N-ethyl-maleimide) but sensitive to 200 microM vanadate, and has similar nucleotide-dependent microtubule binding properties to those of kinesins purified from mammals, sea urchin and Drosophila. This kinesin-like molecule from Dictyostelium, however, is immunologically distinct from bovine and squid neuronal kinesins and supports microtubule movement on glass at four-fold greater velocities (2.0 versus 0.5 microns/sec). Furthermore, AMP-PNP (adenylyl imidodiphosphate), which promotes attachment of previously characterized kinesins to microtubules, decreases the affinity of the Dictyostelium kinesin homolog for microtubules. Thus, an AMP-PNP-induced rigor binding may not be a characteristic of kinesins from lower eukaryotes.     

Microtubule associated protein WAVE DAMPENED2-LIKE (WDL) controls microtubule bundling and the stability of the site of tip-growth in Marchantia polymorpha rhizoids

Tip-growth is a mode of polarized cell expansion where incorporation of new membrane and wall is stably restricted to a single, small domain of the cell surface resulting in the formation of a tubular projection that extends away from the body of the cell. The organization of the microtubule cytoskeleton is conserved among tip-growing cells of land plants: bundles of microtubules run longitudinally along the non-growing shank and a network of fine microtubules grow into the apical dome where growth occurs. Together, these microtubule networks control the stable positioning of the growth site at the cell surface. This conserved dynamic organization is required for the spatial stability of tip-growth, as demonstrated by the formation of sinuous tip-growing cells upon treatment with microtubule-stabilizing or microtubule-destabilizing drugs. Microtubule associated proteins (MAPs) that either stabilize or destabilize microtubule networks are required for the maintenance of stable tip-growth in root hairs of flowering plants. NIMA RELATED KINASE (NEK) is a MAP that destabilizes microtubule growing ends in the apical dome of tip-growing rhizoid cells in the liverwort Marchantia polymorpha. We hypothesized that both microtubule stabilizing and destabilizing MAPs are required for the maintenance of the stable tip-growth in liverworts. To identify genes encoding microtubule-stabilizing and microtubule-destabilizing activities we generated 120,000 UV-B mutagenized and 336,000 T-DNA transformed Marchantia polymorpha plants and screened for defective rhizoid phenotypes. We identified 119 mutants and retained 30 mutants in which the sinuous rhizoid phenotype was inherited. The 30 mutants were classified into at least 4 linkage groups. Characterisation of two of the linkage groups showed that MAP genes–WAVE DAMPENED2-LIKE (WDL) and NIMA-RELATED KINASE (NEK)–are required to stabilize the site of tip growth in elongating rhizoids. Furthermore, we show that MpWDL is required for the formation of a bundled array of parallel and longitudinally orientated microtubules in the non-growing shank of rhizoids where MpWDL-YFP localizes to microtubule bundles. We propose a model where the opposite functions of MpWDL and MpNEK on microtubule bundling are spatially separated and promote tip-growth spatial stability.     

Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.