Tsr-GFP accumulates linearly with time at cell poles, and can be used to differentiate 'old' versus 'new' poles, in Escherichia coli

In Escherichia coli , the chemotaxis receptor protein Tsr localizes abundantly to cell poles. The current study, utilizing a Tsr–GFP fusion protein and time-lapse fluorescence microscopy of individual cell lineages, demonstrates that Tsr accumulates approximately linearly with time at the cell poles and that, in consequence, more Tsr is present at the old pole of each cell than at its newborn pole. The rate of pole-localized Tsr accumulation is large enough that old and new poles can always be reliably distinguished, even for cells whose old poles have had only one generation to accumulate signal. Correspondingly, Tsr–GFP can be reliably used to assign new and old poles to any cell without use of information regarding pole heritage, thus providing a useful tool to analyse cells whose prior history is not available. The absolute level of Tsr–GFP at the old pole of a cell also provides a rough estimate of pole (and thus cell) age.     

TPX2, A novel xenopus MAP involved in spindle pole organization

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.     

Chromosome movement in mitosis requires microtubule anchorage at spindle poles

Anchorage of microtubule minus ends at spindle poles has been proposed to bear the load of poleward forces exerted by kinetochore-associated motors so that chromosomes move toward the poles rather than the poles toward the chromosomes. To test this hypothesis, we monitored chromosome movement during mitosis after perturbation of nuclear mitotic apparatus protein (NuMA) and the human homologue of the KIN C motor family (HSET), two noncentrosomal proteins involved in spindle pole organization in animal cells. Perturbation of NuMA alone disrupts spindle pole organization and delays anaphase onset, but does not alter the velocity of oscillatory chromosome movement in prometaphase. Perturbation of HSET alone increases the duration of prometaphase, but does not alter the velocity of chromosome movement in prometaphase or anaphase. In contrast, simultaneous perturbation of both HSET and NuMA severely suppresses directed chromosome movement in prometaphase. Chromosomes coalesce near the center of these cells on bi-oriented spindles that lack organized poles. Immunofluorescence and electron microscopy verify microtubule attachment to sister kinetochores, but this attachment fails to generate proper tension across sister kinetochores. These results demonstrate that anchorage of microtubule minus ends at spindle poles mediated by overlapping mechanisms involving both NuMA and HSET is essential for chromosome movement during mitosis.     

Inertial force as a possible factor in mitosis

Some previous studies of cell division have suggested that chromosome movements in mitosis involve two distinct forces: one which pulls chromosomes poleward by means of attached fibers, and another which tends to push chromosome arms away from the pole. The latter force may also be a factor in non-chromosomal spindle transport, by which objects other than chromosomes are transported toward or away from spindle poles. Based on a survey of previous literature, this paper makes a prima facie case for describing this latter force as "inertial", since in some respects it can be simulated by centrifugation. A theoretical analysis demonstrates that an inertial force could arise in the spindle from postulated high-frequency, small-amplitude oscillations, which could be caused by changes in coherently processing electron spin alignments at the spindle poles. Some possible experimental approaches to the problem are briefly outlined.     

Kinetochore capture and bi-orientation on the mitotic spindle

Kinetochores are large protein complexes that are formed on chromosome regions known as centromeres. For high-fidelity chromosome segregation, kinetochores must be correctly captured on the mitotic spindle before anaphase onset. During prometaphase, kinetochores are initially captured by a single microtubule that extends from a spindle pole and are then transported poleward along the microtubule. Subsequently, microtubules that extend from the other spindle pole also interact with kinetochores and, eventually, each sister kinetochore attaches to microtubules that extend from opposite poles - this is known as bi-orientation. Here we discuss the molecular mechanisms of these processes, by focusing on budding yeast and drawing comparisons with other organisms.     

Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.     

Intracellular transport of bile acids

Bile acids originate from the liver and are transported via bile to the intestines where they perform an important role in the absorption of lipids and lipid-soluble nutrients. Most of the bile acids are reclaimed from the terminal ileum and returned to the liver via portal blood for reuse. The transport of bile acids is vectorial in both liver and intestinal cells, originating and terminating at opposite poles. Bile acids enter through the basolateral pole in liver cells, and through the apical pole in intestinal cells. During the past decade, much has been learned about the mechanisms by which bile acids enter and exit liver and intestinal cells. By contrast, the mechanisms by which bile acids are transported across cells remain poorly understood. The current body of evidence suggests that bile acids do not traverse the cell by vesicular transport. Although a carrier-mediated mechanism is a likely alternative, only a handful of intracellular proteins capable of binding bile acids have been described. The significance of these proteins in the intracellular transport of bile acids remains to be tested.     

ADY1, a novel gene required for prospore membrane formation at selected spindle poles in Saccharomyces cerevisiae

ADY1 is identified in a genetic screen for genes on chromosome VIII of Saccharomyces cerevisiae that are required for sporulation. ADY1 is not required for meiotic recombination or meiotic chromosome segregation, but it is required for the formation of four spores inside an ascus. In the absence of ADY1, prospore formation is restricted to mainly one or two spindle poles per cell. Moreover, the two spores in the dyads of the ady1 mutant are predominantly nonsisters, suggesting that the proficiency to form prospores is not randomly distributed to the four spindle poles in the ady1 mutant. Interestingly, the meiosis-specific spindle pole body component Mpc54p, which is known to be required for prospore membrane formation, is localized predominantly to only one or two spindle poles per cell in the ady1 mutant. A partially functional Myc-Pfs1p is localized to the nucleus of mononucleate meiotic cells but not to the spindle pole body or prospore membrane. These results suggest that Pfs1p is specifically required for prospore formation at selected spindle poles, most likely by ensuring the functionality of all four spindle pole bodies of a cell during meiosis II.     

Centrosomes as signalling centres

Centrosomes—as well as the related spindle pole bodies (SPBs) of yeast—have been extensively studied from the perspective of their microtubule-organizing roles. Moreover, the biogenesis and duplication of these organelles have been the subject of much attention, and the importance of centrosomes and the centriole–ciliary apparatus for human disease is well recognized. Much less developed is our understanding of another facet of centrosomes and SPBs, namely their possible role as signalling centres. Yet, many signalling components, including kinases and phosphatases, have been associated with centrosomes and spindle poles, giving rise to the hypothesis that these organelles might serve as hubs for the integration and coordination of signalling pathways. In this review, we discuss a number of selected studies that bear on this notion. We cover different processes (cell cycle control, development, DNA damage response) and organisms (yeast, invertebrates and vertebrates), but have made no attempt to be comprehensive. This field is still young and although the concept of centrosomes and SPBs as signalling centres is attractive, it remains primarily a concept—in need of further scrutiny. We hope that this review will stimulate thought and experimentation.     

Cytoplasmic dynein's mitotic spindle pole localization requires a functional anaphase-promoting complex, gamma-tubulin, and NUDF/LIS1 in Aspergillus nidulans

In Aspergillus nidulans, cytoplasmic dynein and NUDF/LIS1 are found at the spindle poles during mitosis, but they seem to be targeted to this location via different mechanisms. The spindle pole localization of cytoplasmic dynein requires the function of the anaphase-promoting complex (APC), whereas that of NUDF does not. Moreover, although NUDF's localization to the spindle poles does not require a fully functional dynein motor, the function of NUDF is important for cytoplasmic dynein's targeting to the spindle poles. Interestingly, a gamma-tubulin mutation, mipAR63, nearly eliminates the localization of cytoplasmic dynein to the spindle poles, but it has no apparent effect on NUDF's spindle pole localization. Live cell analysis of the mipAR63 mutant revealed a defect in chromosome separation accompanied by unscheduled spindle elongation before the completion of anaphase A, suggesting that gamma-tubulin may recruit regulatory proteins to the spindle poles for mitotic progression. In A. nidulans, dynein is not apparently required for mitotic progression. In the presence of a low amount of benomyl, a microtubule-depolymerizing agent, however, a dynein mutant diploid strain exhibits a more pronounced chromosome loss phenotype than the control, indicating that cytoplasmic dynein plays a role in chromosome segregation.     

Microtubule dynamics and chromosome motion visualized in living anaphase cells

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.     

Lesser Prairie-Chicken Survival in Varying Densities of Energy Development

Anthropogenic features increasingly affect ecological processes with increasing human demand for natural resources. Such effects also have the potential to vary depending on the sex and age of an individual because of inherent behavioral and life experience differences. For the lesser prairie-chicken (Tympanuchus pallidicinctus), studies on male survival are limited because most previous research has been focused on females. To better understand patterns of lesser prairie-chicken survival in habitat with varying levels of anthropogenic infrastructure associated with oil and natural gas development, we monitored survival of 178 radio-tagged male and female lesser prairie-chickens in eastern New Mexico, USA, from 2013 to 2015. We examined the relationships of shrub cover, proximity to and density of anthropogenic features (i.e., utility poles), displacement of natural vegetation by anthropogenic features (i.e., area of roads and well pads), and individual demographics (i.e., sex, age) with lesser prairie-chicken survival. Furthermore, we categorized the probable cause of mortality and examined its relationship with oil and gas development intensity (indexed by utility pole density) within 1,425 m of an individual's mortality site or final observed location. We predicted that survival would be lower for individuals exposed to greater levels of anthropogenic features, and that males and subadults would be more negatively affected than females and adults because of increased exposure to predators during the lekking season and naiveté. Relationships between survival and utility pole density, sex, and age were supported in our top-ranked models, whereas models including other anthropogenic and natural features (i.e., roads, well pads, shrub cover) received little support. We predicted a substantial decrease in adult and subadult male survival with increasing densities of utility poles. The relationship between survival and utility pole density for females was weaker and not as clearly supported as for males. We did not find a detectable difference in utility pole counts among probable mortality causes. Our findings highlight the importance of including male lesser prairie-chickens in research and conservation planning, and the negative effect that high densities of anthropogenic features can have on lesser prairie-chicken survival. © 2021 The Wildlife Society.     

Formation of spindle poles by dynein/dynactin-dependent transport of NuMA

NuMA is a large nuclear protein whose relocation to the spindle poles is required for bipolar mitotic spindle assembly. We show here that this process depends on directed NuMA transport toward microtubule minus ends powered by cytoplasmic dynein and its activator dynactin. Upon nuclear envelope breakdown, large cytoplasmic aggregates of green fluorescent protein (GFP)-tagged NuMA stream poleward along spindle fibers in association with the actin-related protein 1 (Arp1) protein of the dynactin complex and cytoplasmic dynein. Immunoprecipitations and gel filtration demonstrate the assembly of a reversible, mitosis-specific complex of NuMA with dynein and dynactin. NuMA transport is required for spindle pole assembly and maintenance, since disruption of the dynactin complex (by increasing the amount of the dynamitin subunit) or dynein function (with an antibody) strongly inhibits NuMA translocation and accumulation and disrupts spindle pole assembly.     

P190RhoGAP prevents mitotic spindle fragmentation and is required to activate Aurora A kinase at acentriolar poles

Assembly of the mitotic spindle is essential for proper chromosome segregation during mitosis. Maintenance of spindle poles requires precise regulation of kinesin- and dynein-generated forces, and improper regulation of these forces disrupts pole integrity leading to pole fragmentation. The formation and function of the mitotic spindle are regulated by many proteins, including Aurora A kinase and the motor proteins Kif2a and Eg5. Here, we characterize a surprising role for the RhoA GTPase-activating protein, p190RhoGAP, in regulating the mitotic spindle. We show that cells depleted of p190RhoGAP arrest for long periods in mitosis during which cells go through multiple transitions between having bipolar and multipolar spindles. Most of the p190RhoGAP-depleted cells finally achieve a stable bipolar attachment and proceed through anaphase. The multipolar spindle phenotype can be rescued by low doses of an Eg5 inhibitor. Moreover, we show that p190RhoGAP-depleted multipolar cells localize Aurora A to all the poles, but the kinase is only activated at the two centriolar poles. Overall, our data identify an unappreciated connection between p190RhoGAP and the proteins that control spindle poles including Aurora A kinase and Eg5 that is required to prevent or correct spindle pole fragmentation.     

Attaching to spindles before they form: Do early incorrect chromosome-microtubule attachments promote meiotic segregation fidelity?

The proper partitioning of the genome during meiosis depends on the correct segregation of chromosomes. Errors in this process result in the production of aneuploid gametes, a major cause of birth defects and infertility in humans. In order to segregate properly in meiosis, homologous chromosome partners must attach to microtubules that emanate from opposites poles of the spindle. However, a recent study in yeast has shown that, remarkably, the initial attachments between microtubules and the chromosomes are usually incorrect, which would lead to catastrophic segregation errors, but they are nearly always corrected through the detachment and reattachment of the microtubules. Here we review the reasons for the initial incorrect attachments, which stem from the timing of their formation early in the spindle assembly process, and the fact that the microtubule organizers, called spindle pole bodies in yeast, are not equal. One spindle pole body is older and better able to produce microtubules that attach to the chromosomes. We draw parallels to recent findings in animal cells and suggest that these early microtubule attachments, while often incorrect, may serve an important role in spindle assembly, which, in the long-term, promotes high-fidelity chromosome segregation.     

Cotranslational transport of ABP140 mRNA to the distal pole of S. cerevisiae

In budding yeast, several mRNAs are selectively transported into the daughter cell in an actin-dependent manner by a specialized myosin system, the SHE machinery. With ABP140 mRNA, we now describe the first mRNA that is transported in the opposite direction and localizes to the distal pole of the mother cell, independent of the SHE machinery. Distal pole localization is not observed in mutants devoid of actin cables and can be disrupted by latrunculin A. Furthermore, localization of ABP140 mRNA requires the N-terminal actin-binding domain of Abp140p to be expressed. By replacing the N-terminal localization motif, ABP140 mRNA can be retargeted to different subcellular structures. In addition, accumulation of the mRNA at the distal pole can be prevented by disruption of polysomes. Using the MS2 system, the mRNA was found to associate with actin cables and to follow actin cable dynamics. We therefore propose a model of translational coupling, in which ABP140 mRNA is tethered to actin cables via its nascent protein product and is transported to the distal pole by actin retrograde flow.     

Global Motion Percept Mediated through Integration of Barber Poles Presented in Bilateral Visual Hemifields

How is motion information that has been obtained through multiple viewing apertures integrated to form a global motion percept? We investigated the mechanisms of motion integration across apertures in two hemifields by presenting gratings through two rectangles (that form the dual barber poles) and recording the perceived direction of motion by human observers. To this end, we presented dual barber poles in conditions with various inter-component distances between the apertures and evaluated the degree to which the hemifield information was integrated by measuring the magnitude of the perceived barber pole illusion. Surprisingly, when the inter-component distance between the two apertures was short, the perceived direction of motion of the dual barber poles was similar to that of a single barber pole formed by the concatenation of the two component barber poles, indicating motion integration is achieved through a simple concatenation mechanism. We then presented dual barber poles in which the motion and contour properties of the two component barber poles differed to characterize the constraints underlying cross-hemifield integration. We found that integration is achieved only when phase, speed, wavelength, temporal frequency, and duty cycle are identical in the two barber poles, but can remain robust when the contrast of the two component barber poles differs substantially. We concluded that a motion stimulus presented in bilateral hemifields tends to be integrated to yield a global percept with a substantial tolerance for spatial distance and contrast difference.     

Is Escherichia coli getting old?

Whether or not bacteria divide symmetrically, the inheritance of cell poles is always asymmetrical. Because each cell carries an old and a new pole, its daughters will not be the same. Tracking poles of cells and measuring their lengths and doubling times in micro-colonies, Stewart et al.1 observed that growth rate diminished in cells inheriting old poles and concluded that these cells are susceptible to aging. Here, their results are compared with studies on the variabilities of length and age at division. It is argued that the decreased growth rate in old pole cells falls within the expected variation and may therefore be sufficiently far from a catastrophe-like cell death through aging.     

Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication.

Aneuploidy and polyploidy are commonly observed in transformed cells. These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome. The centrosome is the organizing center for the mitotic spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the spindle pole body. We review how defects in spindle pole body duplication or function lead to genetic instability in yeast. There are several well documented instances of genetic instability in yeast that can be traced to the spindle pole body, all of which serve as models for genetic instability in transformed cells.