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The use of microtechnique for studying cell division is well established (BeggEllis, 1979; Wadsworth, 1999; ZhangNicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

The use of microtechnique for studying cell division is well established (Begg & Ellis, 1979; Wadsworth, 1999; Zhang & Nicklas, 1999). The advantage of microinjection in cell division research is the timed delivery of a macromolecules at a particular stage of mitosis (for example, pre- vs postanaphase), which can circumvent the spindle assembly checkpoint (Hinchcliffe et al., 2016). Micromanipulation can be used to remove whole organelles, such as the centrosome or nucleus and examine the effects on cell division (Hinchcliffe et al., 2001; Hornick et al., 2011). The focus of this chapter is on methods for microinjection and micromanipulation of cultured mammalian cells. We describe pulling and shaping microneedles, as well as the imaging chambers we use. We also provide information on cell culture conditions, and imaging techniques used for our long-term observation studies, which allow cells to be followed on the order of several days.

In vertebrate somatic cells the centrosome functions as the major microtubule-organizing center (MTOC), which splits and separates to form the poles of the mitotic spindle. However, the role of the centriole-containing centrosome in the formation of bipolar mitotic spindles continues to be controversial. Cells normally containing centrosomes are still able to build bipolar spindles after their centrioles have been removed or ablated. In naturally occurring cellular systems that lack centrioles - such as plant cells and many oocytes - bipolar spindles form in the complete absence of canonical centrosomes. These observations have led to the notion that centrosomes play no role during mitosis. However, recent work has re-examined spindle assembly in the absence of centrosomes, both in cells that naturally lack them, and those that have had them experimentally removed. The results of these studies suggest that an appreciation of microtubule network organization- both before and after nuclear envelope breakdown (NEB) - is the key to understanding the mechanisms that regulate spindle assembly and the generation of bipolarity.

SummaryThe role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1–3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays—termed an amphiaster (“a star on both sides” [4])—that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ∼35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP—the small GTPase Ran in its GTP bound state—could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis.

The assembly of a bipolar spindle lies at the heart of mitotic chromosome segregation. In animal somatic cells, the process of spindle assembly involves multiple complex interactions between various cellular compartments, including an emerging antiparallel microtubule network, microtubule-associated motor proteins and spindle assembly factors, the cell's cortex, and the chromosomes themselves. The result is a dynamic structure capable of aligning pairs of sister chromatids, sensing chromosome misalignment, and generating force to segregate the replicated genome into two daughters. Because the centrosome lies at the center of the array of microtubule minus-ends, and the essential one-to-two duplication of the centrosome prior to mitosis is linked to cell cycle progression, this organelle has long been implicated as a device to generate spindle bipolarity. However, this classic model for spindle assembly is challenged by observations and experimental manipulations demonstrating that acentrosomal cells can and do form bipolar spindles, both mitotic and meiotic. Indeed, recent comprehensive proteomic analysis of centrosome-dependent versus independent mitotic spindle assembly mechanisms reveals a large, common set of genes required for both processes, with very few genes needed to differentiate between the two. While these studies cast doubt on an absolute role for the centrosome in establishing spindle polarity, it is clear that having too few or too many centrosomes results in abnormal chromosome segregation and aneuploidy. Here we review the case both for and against the role of the centrioles and centrosomes in ensuring proper assembly of a bipolar spindle, an essential element in the maintenance of genomic stability.

Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing γ-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures—distinct from elements of the microtubule-organizing center—that is required for the somatic cell cycle to progress through G 1 into S phase. Once the cell is in S phase, these core structures are not needed for the G 2 -M phase transition.

The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2–cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.

The centrosome plays an important role in regulating cell cycle progression during interphase and mitosis, but the underlying mechanisms of regulation are unclear. A recent study, which has identified a rare centriolar polypeptide, may provide part of the answer.

When CHO cells are arrested in S-phase, they undergo repeated rounds of centrosome duplication without cell-cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S-phase for up to 72 h, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid. We find that in such cells, the pre-existing centrosomes remain, and a variable number of foci—containing α/γ-tubulin and centrin 2—assemble at the nuclear periphery. When the colcemid is washed out, the nuclear-associated foci disappear, and cells assemble new centriole-containing centrosomes, which accumulate the centriole scaffold protein SAS-6, nucleate microtubule asters, and form functional mitotic spindle poles. The number of centrosomes that assemble following colcemid washout increases with duration of S-phase arrest, even though the number of nuclear-associated foci or pre-existing centrosomes does not increase. This suggests that during S-phase, a cryptic generative event occurs repeatedly, even in the absence of new triplet microtubule assembly. When triplet microtubule assembly is restored, these cryptic generative events become realized, and multiple centriole-containing centrosomes assemble. J. Cell. Physiol. 225: 454–465, 2010. © 2010 Wiley-Liss, Inc.

Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.

The single interphase centrosome duplicates exactly once in a cell cycle dependent fashion, yielding two daughter centrosomes, each of which contributes to the assembly of a mitotic spindle pole (reviewed in Stearns, 2001; Delattre and Gonczy, 2004). The two initial events in the centrosome duplication cycle are commonly thought to be the “disorientation” or “disengagement” of the parental centriole pair (termed the diplosome), which prepares the centrosome for duplication, followed by the assembly of short daughter centrioles—called pro-centrioles—at right angles to the pre-existing centrioles (Kuriyama and Borisy, 1981; Kochanski and Borisy, 1990). Centrosome duplication is coordinated with nuclear events during cell cycle progression (reviewed in Hinchcliffe and Sluder, 2001a). This is important because centrosomes play a dominant role in spindle pole organization; there must be two and only two centrosomes present as the cell enters mitosis, otherwise the potential exists to assemble a multipolar spindle, resulting in an increased frequency of aneuploidy and tumor cell progression (reviewed in Brinkley, 2001; Fisk et al., 2002; Sluder and Nordberg, 2004). Understanding the cell cycle control of centrosome duplication has been difficult, primarily because there is no good marker to signify when duplication begins. By the time morphologically distinct centrioles have formed, and are recognizable in the electron microscope, the initial events of centrosome duplication may have already taken place (discussed in Hinchcliffe and Sluder, 1998). In order to examine the cell cycle regulation of centrosome duplication, several studies have relied on arresting cell cycle progression in a particular phase, and then assaying whether or not centrosomes are capable of duplicating one or more times (reviewed in Hinchcliffe and Sluder, 2001a). The results of this work have experimentally defined those stages that are capable of supporting centrosome duplication, and those that cannot. In cells that are driven into G0, the centrosome does not duplicate until serum-released (Tucker et al., 1979; Okuda et al., 2000). During S-phase arrest, centrosomes can clearly undergo repeated rounds of duplication in both zygotes and certain transformed somatic cells (Kuriyama et al., 1986; Sluder and Lewis, 1987; Raff and Glover, 1988; Balczon et al., 1995; Hinchcliffe et al., 1998). In cells arrested in G2 with topoisomerase inhibitors, the duplicated centrosome cannot undergo further rounds of duplication (Balczon et al., 1995). When mitosis is prolonged in a variety of cell types, centrosome duplication cannot proceed (Hinchcliffe et al., 1998; Vidwans et al., 1999), although the diplosome can undergo disengagement (Tsou and Stearns, 2006). While the aforementioned studies have provided information about which cell cycle stages can or cannot support centrosome duplication, there are conflicting reports about whether or not centrosome duplication can occur during G1. This phase of the cell cycle is particularly important, because centrosome duplication is said to normally occur at the G1/S phase transition (Robbins et al., 1968; Rattner and Phillips, 1973; Kuriyama and Borisy, 1981; Vorobjev and Chentsov, 1982; Alvey, 1985). The implication is that as the cell cycle proceeds into S-phase, the signals that initiate DNA replication also drive the duplication of the centrosome (discussed in Sluder and Rieder, 1996). However, it remains a formal possibility that centrosome duplication could be initiated without the activation of these signals, and then continues in parallel as the cell cycle transitions into S-phase. Several studies have suggested that centrosome duplication cannot occur until after the G1/S phase transition (Robbins et al., 1968; Vorobjev and Chentsov, 1982; Marshall et al., 2001), while other have revealed that it can proceed prior to entry into S-phase (Rattner and Phillips, 1973; Winey and Byers, 1993; Fukasawa et al., 1996; Schutz et al., 1997; Hinchcliffe et al., 1998; Uzawa et al., 2004). However, many of these studies have examined when centrosome duplication occurs in cycling cells, rather than examining the effect of prolonging the cell cycle in G1, and assaying for centrosome duplication. Put another way, centrosome duplication may begin in G1, and by the time morphologically distinct centrioles are recognizable in the electron microscope, the cell cycle has proceeded into S-phase. The consequences of prolonging G1 on the centrosome duplication cycle have not been experimentally tested in mammalian somatic cells. To address this, we use both transformed and non-transformed cultured Chinese hamster cells to directly test whether or not centriole reproduction can occur when the cell cycle is prolonged in G1.

The essence of successful mitosis is the generation of two genetically identical daughter cells. This requires the assembly of a strictly bipolar mitotic apparatus that will ensure that all daughter chromosomes are segregated to opposite sides of the cell before the completion of mitosis. The agent of the higher animal cell for the establishment of this essential “twoness” is the pair of centrosomes that organize the spindle poles during mitosis (Mazia 1987). Before the cell enters mitosis, the single interphase centrosome duplicates or reproduces exactly once, and the daughter centrosomes form the two poles of the spindle after breakdown of the nuclear envelope. Failure of the cell to precisely control this duplication of the centrosome can have disastrous consequences. If the centrosome fails to duplicate before the onset of mitosis, most somatic cells eventually return to interphase without dividing, which causes polyploidy. If the centrosome duplicates more than once in a cell cycle, then a multipolar spindle may be assembled and the chromosomes may be unequally distributed to the daughter cells, leading to genetic imbalances that produce cells with aggressive growth characteristics (for reviews, see Orr-Weaver and Weinberg 1998; Brinkley 2001). In fact, the cells of many lethal human tumors are genetically unstable and have abnormally high numbers of centrosomes (Lingle et al. 1998; Pihan et al. 1998; Carroll et al. 1999). Extra centrosomes are a problem because the condition is not remediated; cells do not have a checkpoint that aborts mitosis in response to extra spindle poles (Sluder et al. 1997). In this review, we focus on the mechanisms that control centrosome duplication and coordinate it with nuclear events during the cell cycle.

Publisher Summary Cytoplasmic extracts have been used to investigate certain aspects of the centrosome reproductive cycle. It is the ability to assemble multiple centrosomes without cell cycle progression that makes egg cytoplasm such an attractive system to study the regulation of the centrosome duplication cycle. This chapter describes development of an extract that would support repeated rounds of centrosome reproduction. For this purpose, Xenopus extract seeded with demembranated sperm heads is cycled. These interphase extracts are then treated with the DNA synthesis inhibitor aphidicolin, which activates the checkpoint that monitors the completion of DNA synthesis, leading to a cell cycle arrest in S phase. Video microscopy, combined with polarization optics, is used to follow centrosome duplication events in real time. It is found that such extracts recapitulated the repeated rounds of centrosome reproduction seen in living zygotes. This chapter details the crafting and use of these S-phase-arrested egg extracts for the study of centrosome reproduction.

Publisher Summary The centrosome is the ensemble of structures that nucleates the interphase array of cytoplasmic microtubles and assembles the poles of the mitotic spindle. This chapter discusses the events and controls for centrosome reproduction. Coordination of centrosome reproduction with nuclear events in the cell cycle is also discussed. The sequence of the events of centrosome reproduction is (1) splitting, (2) disorientation, (3) duplication, (4) licensing, (5) disjunction, and (6) separation. The controls for centrosome reproduction are carried by limits that are intrinsic to the centrosome or by extrinsic controls imposed by changing cytoplasmic conditions. The cycle of centrosome reproduction can occur repeatedly in the absence of cell-cycle progression. One way of investigating the coordination of centrosome reproduction and nuclear events in the cell cycle is to characterize which cell-cycle stages support centrosome reproduction and which do not. The approach used is to arrest cells in a particular phase of the cell cycle and then determine if the centrosome reproduces one or more times without further experimental intervention. The sequential activation and inactivation of one or more of the cyclin-dependent kinases (Cdks) could function as the cytoplasmic mechanism that coordinately drives centrosome duplication and nuclear events in the cell cycle.

Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3–4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.

Despite over one hundred years of research, the duplication of the centrosome is a poorly understood process. Three recent papers — exploring three different kinases — may have provided the answer.