Interphase Microtubule Arrays Research Articles

CSPP1 stabilizes growing microtubule ends and damaged lattices from the luminal side.

Microtubules are dynamic cytoskeletal polymers, and their organization and stability are tightly regulated by numerous cellular factors. While regulatory proteins controlling the formation of interphase microtubule arrays and mitotic spindles have been extensively studied, the biochemical mechanisms responsible for generating stable microtubule cores of centrioles and cilia are poorly understood. Here, we used in vitro reconstitution assays to investigate microtubule-stabilizing properties of CSPP1, a centrosome and cilia-associated protein mutated in the neurodevelopmental ciliopathy Joubert syndrome. We found that CSPP1 preferentially binds to polymerizing microtubule ends that grow slowly or undergo growth perturbations and, in this way, resembles microtubule-stabilizing compounds such as taxanes. Fluorescence microscopy and cryo-electron tomography showed that CSPP1 is deposited in the microtubule lumen and inhibits microtubule growth and shortening through two separate domains. CSPP1 also specifically recognizes and stabilizes damaged microtubule lattices. These data help to explain how CSPP1 regulates the elongation and stability of ciliary axonemes and other microtubule-based structures.

Overlapping Localization of MAP65-2, -6, and -7 in Arabidopsis Hypocotyl Cells.

Microtubules are essential components of eukaryotic cells. Myriad proteins associate with microtubules to facilitate the organization and operation of microtubule arrays. Various M icrotubule A ssociated P roteins (MAPs) assist the assembly and function of mitotic spindles and interphase arrays. Nine MAP65 genes exist in the genome of the acentrosomal model plant, Arabidopsis thaliana, and the function of majority of these proteins is unclear. To address this knowledge gap, we demonstrate the localization of A. thaliana MAP65-6 and MAP65-7 fusion proteins expressed from native promoters in interphase cells of developing A. thaliana seedlings. Analyses of these fusion proteins co-expressed with alpha-tubulin 6 reporters indicate that MAP65-6 and MAP65-7 bind a subset of interphase microtubules. Co-expression of GFP: MAP65-6 with mCherry: MAP65-2 from native promoters in A. thaliana showed overlapping localization patterns on interphase microtubule bundles. Collectively, these data suggested that MAP65-2 , -6, and -7 bind cortical microtubule bundles in plant interphase microtubule arrays.

Super-Resolution Microscopy and FIB-SEM Imaging Reveal Parental Centriole-Derived, Hybrid Cilium in Mammalian Multiciliated Cells.

Motile cilia are cellular beating machines that play a critical role in mucociliary clearance, cerebrospinal fluid movement, and fertility. In the airways, hundreds of motile cilia present on the surface of a multiciliated epithelia cell beat coordinately to protect the epithelium from bacteria, viruses, and harmful particulates. During multiciliated cell differentiation, motile cilia are templated from basal bodies, each extending a basal foot-an appendage linking motile cilia together to ensure coordinated beating. Here, we demonstrate that among the many motile cilia of a multiciliated cell, a hybrid cilium with structural features of both primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells, originates from parental centrioles, and its cellular position is biased and dependent on ciliary beating. Furthermore, we show that the hybrid cilium emerges independently of other motile cilia and functions in regulating basal body alignment.

Reorganization of paclitaxel-stabilized microtubule arrays at mitotic entry: roles of depolymerizing kinesins and severing proteins

ABSTRACTPaclitaxel is a widely used anti-cancer treatment that disrupts cell cycle progression by blocking cells in mitosis. The block at mitosis, with spindles assembled from short microtubules, is surprising given paclitaxel’s microtubule stabilizing activity and the need to depolymerize long interphase microtubules prior to spindle formation. Cells must antagonize paclitaxel’s microtubule stabilizing activity during a brief window of time at the transition from interphase to mitosis, allowing microtubule reorganization into a mitotic spindle, although the mechanism underlying microtubule depolymerization in the presence of paclitaxel has not been examined. Here we test the hypothesis that microtubule severing and/or depolymerizing proteins active at mitotic entry are necessary to clear the interphase array in paclitaxel-treated cells and allow subsequent formation of mitotic spindles formed of short microtubules. A549 and LLC-PK1 cells treated with 30nM paclitaxel approximately 4 h prior to mitotic entry successfully progress through the G2/M transition by clearing the interphase microtubule array from the cell interior outward to the cell periphery, a spatial pattern of reorganization that differs from that of cells possessing dynamic microtubules. Depletion of kinesin-8s, KIF18A and/or KIF18B obstructed interphase microtubule clearing at mitotic entry in paclitaxel-treated cells, with KIF18B making the larger contribution. Of the severing proteins, depletion of spastin, but not katanin, reduced microtubule loss as cells entered mitosis in the presence of paclitaxel. These results support a model in which KIF18A, KIF18B, and spastin promote interphase microtubule array disassembly at mitotic entry and can overcome paclitaxel-induced microtubule stability specifically at the G2/M transition.

Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum.

The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.

Opposing kinesin complexes queue at plus tips to ensure microtubule catastrophe at cell ends.

In fission yeast, the lengths of interphase microtubule (iMT) arrays are adapted to cell length to maintain cell polarity and to help centre the nucleus and cell division ring. Here, we show that length regulation of iMTs is dictated by spatially regulated competition between MT‐stabilising Tea2/Tip1/Mal3 (Kinesin‐7) and MT‐destabilising Klp5/Klp6/Mcp1 (Kinesin‐8) complexes at iMT plus ends. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus ends of iMT bundles, thereby restricting access to the plus ends by Klp5/Klp6/Mcp1, which accumulate behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus ends and MT catastrophe. These data show that in vivo, whilst an iMT length‐dependent model for catastrophe factor accumulation has validity, length control of iMTs is an emergent property reflecting spatially regulated competition between distinct kinesin complexes at the MT plus tip.

Centrosomes are multifunctional regulators of genome stability

The maintenance of genome stability is critical for proper cell function, and loss of this stability contributes to many human diseases and developmental disorders. Therefore, cells have evolved partially redundant mechanisms to monitor and protect the genome. One subcellular organelle implicated in the maintenance of genome stability is the centrosome, best known as the primary microtubule organizing center of most animal cells. Centrosomes serve many different roles throughout the cell cycle, and many of those roles, including mitotic spindle assembly, nucleation of the interphase microtubule array, DNA damage response, and efficient cell cycle progression, have been proposed to help maintain genome stability. As a result, the centrosome is itself a highly regulated entity. Here, we review evidence concerning the significance of the centrosome in promoting genome integrity. Recent advances permitting acute and persistent centrosome removal suggest we still have much to learn regarding the specific function and actual importance of centrosomes in different contexts, as well as how cells may compensate for centrosome dysfunction to maintain the integrity of the genome. Although many animal cells survive and proliferate in the absence of centrosomes, they do so aberrantly. Based on these and other studies, we conclude that centrosomes serve as critical, multifunctional organelles that promote genome stability.

Organization of microtubule assemblies in Dictyostelium syncytia depends on the microtubule crosslinker, Ase1.

It has long been known that the interphase microtubule (MT) array is a key cellular scaffold that provides structural support and directs organelle trafficking in eukaryotic cells. Although in animal cells, a combination of centrosome nucleating properties and polymer dynamics at the distal microtubule ends is generally sufficient to establish a radial, polar array of MTs, little is known about how effector proteins (motors and crosslinkers) are coordinated to produce the diversity of interphase MT array morphologies found in nature. This diversity is particularly important in multinucleated environments where multiple MT arrays must coexist and function. We initiate here a study to address the higher ordered coordination of multiple, independent MT arrays in a common cytoplasm. Deletion of a MT crosslinker of the MAP65/Ase1/PRC1 family disrupts the spatial integrity of multiple arrays in Dictyostelium discoideum, reducing the distance between centrosomes and increasing the intermingling of MTs with opposite polarity. This result, coupled with previous dynein disruptions suggest a robust mechanism by which interphase MT arrays can utilize motors and crosslinkers to sense their position and minimize overlap in a common cytoplasm.

GCP-WD Mediates γ-TuRC Recruitment and the Geometry of Microtubule Nucleation in Interphase Arrays of Arabidopsis

Many differentiated animal cells, and all higher plant cells, build interphase microtubule arrays of specific architectures without benefit of a central organizer, such as a centrosome, to control the location and geometry of microtubule nucleation. These acentrosomal arrays support essential cell functions such as morphogenesis, but the mechanisms by which the new microtubules are positioned and oriented are poorly understood. In higher plants, nucleation of microtubules arises from distributed γ-tubulin ring complexes (γ-TuRCs) at the cell cortex that are associated primarily with existing microtubules and from which new microtubules are nucleated in a geometrically bimodal fashion, either in parallel to the mother microtubule or as a branching event at a mean angle of approximately 40° to the mother microtubule. By imaging the dynamics of individual nucleation events in Arabidopsis, we found that a conserved peripheral protein of the γ-TuRC, GCP-WD/NEDD1, associated with motile γ-TuRCs and localized to nucleation events. Knockdown of this essential protein resulted in reduction of γ-TuRC recruitment to cortical microtubules and total nucleation frequency, showing that GCP-WD controls γ-TuRC positioning and function in these interphase arrays. Further, we discovered an unexpected role for GCP-WD in determining the geometry of microtubule-dependent microtubule nucleation, where it acts to increase the likelihood of branching over parallel nucleation. Cells with normally complex patterns of cortical array organization constructed simpler arrays with cell-wide ordering, suggesting that control of nucleation frequency, positioning, and geometry by GCP-WD allows plant cells to build alternative cortical array architectures.

Cep192 controls the balance of centrosome and non-centrosomal microtubules during interphase.

Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192’s mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192’s function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

Mzt1/Tam4, a fission yeast MOZART1 homologue, is an essential component of the γ-tubulin complex and directly interacts with GCP3Alp6

In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64-amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3(Alp6). Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3(Alp6).

Centrobin regulates centrosome function in interphase cells by limiting pericentriolar matrix recruitment

The amount of pericentriolar matrix at the centrosome is tightly linked to both microtubule nucleation and centriole duplication, although the exact mechanism by which pericentriolar matrix levels are regulated is unclear. Here we show that Centrobin, a centrosomal protein, is involved in regulating these levels. Interphase microtubule arrays in Centrobin-depleted cells are more focused around the centrosome and are less stable than the arrays in control cells. Centrobin-depleted cells initiate microtubule nucleation more rapidly than control cells and exhibit an increase in the number of growing microtubule ends emanating from the centrosome, while the parameters of microtubule plus end dynamics around the centrosome are not significantly altered. Finally, we show that Centrobin depletion results in the increased recruitment of pericentriolar matrix proteins to the centrosome, including γ-tubulin, AKAP450, Kendrin and PCM-1. We propose that Centrobin might regulate microtubule nucleation and organization by controlling the amount of pericentriolar matrix.

MAP65‐1 and MAP65‐2 promote cell proliferation and axial growth in Arabidopsis roots

We investigated the role of the Arabidopsis microtubule associated proteins 65-1 and 65-2 (MAP65-1 and MAP65-2) in the control of axial root growth. Transgenic plants expressing fluorescent fusion proteins from native promoters indicated exactly overlapping accumulation of MAP65-1 and MAP65-2 in the root tip and elongation zone. Nearly identical protein accumulation patterns were observed when MAP65-1 and MAP65-2 were expressed behind a constitutive CaMV 35S promoter, suggesting a level of post-transcriptional control that restricts these proteins to rapidly growing portions of the root. Co-expression of MAP65-1 and MAP65-2 fusion proteins showed precise co-localization to interphase and cytokinetic microtubule arrays. In interphase root tip cells, the fluorescent protein fusions labeled microtubules that were organized into a variety of different array patterns. In the rapidly growing cells of the root elongation zone, we found MAP65-1 and MAP65-2 co-localized exclusively to the lateral faces of cells that were axially extending. Genetic analysis showed that MAP65-1 and MAP65-2 are coordinately required for proper root elongation. Double map65-1-1 map65-2-2 mutant roots from dark-grown plants contained 50% fewer cells per file than wild-type roots, but we found no evidence that cytokinesis was disrupted. We additionally discovered that cell length was significantly shorter in the mature regions of the root beyond the zone where MAP65-1 and MAP65-2 accumulated. Our data indicate that MAP65-1 and MAP65-2 play a critical role in root growth by promoting cell proliferation and axial extension.

S. pombe kinesins-8 promote both nucleation and catastrophe of microtubules.

The kinesins-8 were originally thought to be microtubule depolymerases, but are now emerging as more versatile catalysts of microtubule dynamics. We show here that S. pombe Klp5-436 and Klp6-440 are non-processive plus-end-directed motors whose in vitro velocities on S. pombe microtubules at 7 and 23 nm s−1 are too slow to keep pace with the growing tips of dynamic interphase microtubules in living S. pombe. In vitro, Klp5 and 6 dimers exhibit a hitherto-undescribed combination of strong enhancement of microtubule nucleation with no effect on growth rate or catastrophe frequency. By contrast in vivo, both Klp5 and Klp6 promote microtubule catastrophe at cell ends whilst Klp6 also increases the number of interphase microtubule arrays (IMAs). Our data support a model in which Klp5/6 bind tightly to free tubulin heterodimers, strongly promoting the nucleation of new microtubules, and then continue to land as a tubulin-motor complex on the tips of growing microtubules, with the motors then dissociating after a few seconds residence on the lattice. In vivo, we predict that only at cell ends, when growing microtubule tips become lodged and their growth slows down, will Klp5/6 motor activity succeed in tracking growing microtubule tips. This mechanism would allow Klp5/6 to detect the arrival of microtubule tips at cells ends and to amplify the intrinsic tendency for microtubules to catastrophise in compression at cell ends. Our evidence identifies Klp5 and 6 as spatial regulators of microtubule dynamics that enhance both microtubule nucleation at the cell centre and microtubule catastrophe at the cell ends.

Patronin Regulates the Microtubule Network by Protecting Microtubule Minus Ends

Tubulin assembles into microtubule polymers that have distinct plus and minus ends. Most microtubule plus ends in living cells are dynamic; the transitions between growth and shrinkage are regulated by assembly-promoting and destabilizing proteins. In contrast, minus ends are generally not dynamic, suggesting their stabilization by some unknown protein. Here, we have identified Patronin (also known as ssp4) as a protein that stabilizes microtubule minus ends in Drosophila S2 cells. In the absence of Patronin, minus ends lose subunits through the actions of the Kinesin-13 microtubule depolymerase, leading to a sparse interphase microtubule array and short, disorganized mitotic spindles. In vitro, the selective binding of purified Patronin to microtubule minus ends is sufficient to protect them against Kinesin-13-induced depolymerization. We propose that Patronin caps and stabilizes microtubule minus ends, an activity that serves a critical role in the organization of the microtubule cytoskeleton.

Unsaturated genistein disaccharide glycoside as a novel agent affecting microtubules

Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic derivative of genistein significantly more cytotoxic than genistein, 7- O-(2,3,4,6-tetra- O-acetyl-β- d-galactopyranosyl)-(1→4)-(6- O-acetyl-hex-2-ene-α- d-erythro-pyranosyl)genistein, shortly named G21. We found that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of the interphase microtubule array.

Ciliary Abnormalities Due to Defects in the Retrograde Transport Protein DYNC2H1 in Short-Rib Polydactyly Syndrome

The short-rib polydactyly (SRP) syndromes are a heterogeneous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth.

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L.

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

A Multicomponent Assembly Pathway Contributes to the Formation of Acentrosomal Microtubule Arrays in InterphaseDrosophilaCells

In animal cells, centrosomes nucleate microtubules that form polarized arrays to organize the cytoplasm. Drosophila presents an interesting paradox however, as centrosome-deficient mutant animals develop into viable adults. To understand this discrepancy, we analyzed behaviors of centrosomes and microtubules in Drosophila cells, in culture and in vivo, using a combination of live-cell imaging, electron microscopy, and RNAi. The canonical model of the cycle of centrosome function in animal cells states that centrosomes act as microtubule-organizing centers throughout the cell cycle. Unexpectedly, we found that many Drosophila cell-types display an altered cycle, in which functional centrosomes are only present during cell division. On mitotic exit, centrosomes disassemble producing interphase cells containing centrioles that lack microtubule-nucleating activity. Furthermore, steady-state interphase microtubule levels are not changed by codepleting both gamma-tubulins. However, gamma-tubulin RNAi delays microtubule regrowth after depolymerization, suggesting that it may function partially redundantly with another pathway. Therefore, we examined additional microtubule nucleating factors and found that Mini-spindles, CLIP-190, EB1, or dynein RNAi also delayed microtubule regrowth; surprisingly, this was not further prolonged when we codepleted gamma-tubulins. Taken together, these results modify our view of the cycle of centrosome function and reveal a multi-component acentrosomal microtubule assembly pathway to establish interphase microtubule arrays in Drosophila.

CDK5RAP2 Is a Pericentriolar Protein That Functions in Centrosomal Attachment of the γ-Tubulin Ring Complex

Microtubule nucleation and organization by the centrosome require gamma-tubulin, a protein that exists in a macromolecular complex called the gamma-tubulin ring complex (gammaTuRC). We report characterization of CDK5RAP2, a novel centrosomal protein whose mutations have been linked to autosomal recessive primary microcephaly. In somatic cells, CDK5RAP2 localizes throughout the pericentriolar material in all stages of the cell cycle. When overexpressed, CDK5RAP2 assembled a subset of centrosomal proteins including gamma-tubulin onto the centrosomes or under the microtubule-disrupting conditions into microtubule-nucleating clusters in the cytoplasm. CDK5RAP2 associates with the gammaTuRC via a short conserved sequence present in several related proteins found in a range of organisms from fungi to mammals. The binding of CDK5RAP2 is required for gammaTuRC attachment to the centrosome but not for gammaTuRC assembly. Perturbing CDK5RAP2 function delocalized gamma-tubulin from the centrosomes and inhibited centrosomal microtubule nucleation, thus leading to disorganization of interphase microtubule arrays and formation of anastral mitotic spindles. Together, CDK5RAP2 is a pericentriolar structural component that functions in gammaTuRC attachment and therefore in the microtubule organizing function of the centrosome. Our findings suggest that centrosome malfunction due to the CDK5RAP2 mutations may underlie autosomal recessive primary microcephaly.