Lastly, and most importantly, random distribution of Golgi membranes is not sufficient to explain their distinct organization that could be tracked to the vicinity of the emerging spindle poles. These results implied the existence of an underlying structure that was involved in regulating Golgi membrane position and dynamics during mitosis. Analyses of microtubules and Golgi markers by confocal immunofluorescence microscopy were consistent with the live-cell data suggesting that early mitotic Golgi membranes were organized around the two pole regions of the emerging mitotic spindle.
Furthermore, mitotic Golgi fragments frequently appeared associated with astral microtubules, both proximal and distal to the spindle, strongly suggesting a role for the microtubules themselves in mitotic Golgi organization.
This hypothesis is supported by the observation that the organization of mitotic Golgi is disrupted after treatment of prometaphase cells with the microtubule disrupting drug nocodazole.
However, additional work is required to determine if the role of microtubules is direct, or rather serves to organize another element of the cytoskeleton that directly regulates Golgi behavior during mitosis. An interaction of mitotic Golgi with elements of the developing mitotic spindle could provide a potential explanation for our observations on the behavior of the Golgi during mitosis, such as the regulated tethering of Golgi membranes, and the organization of prophase Golgi in the future spindle region.
Furthermore, the major period of mitotic Golgi dispersal, during the prophase-metaphase transition, is coincident with an abrupt depolymerization and rearrangement of microtubules to form the mitotic spindle Zhai et al. Therefore, one speculative scenario is that the tethering, organization, and displacement processes are all accomplished by the association of mitotic Golgi with the reorganizing microtubules of the evolving spindle. The failure to observe clear indications of motor protein—dependent movement was consistent with previous reports showing that motor-dependent plus and minus end—directed movement of membranes on microtubules is inhibited during mitosis Allan and Vale, Therefore, in the absence of obvious motor driven displacement, we suggest that the dynamics of the microtubules themselves may provide the force involved in reorganization of mitotic Golgi fragments.
Similarly, association with the growing and shrinking ends of microtubules is sufficient to translocate membranes in vitro, via a tip attachment complex TAC , which may be a source for the tethering and reorganizing force exerted on Golgi membranes during mitosis Waterman-Storer et al.
Through an analysis of mitotic Golgi disassembly in vivo we have been able to provide direct evidence for the existence of an ordered inheritance strategy for the Golgi apparatus during cell division.
Moreover, the experimental findings raise several interesting issues concerning the behavior of Golgi membranes during mitosis. Foremost, why do Golgi membranes disassemble into dispersed tubulo-vesicular clusters during mitosis? The previous belief was that the fragmentation process facilitated a chance-based partitioning, but the organization of mitotic Golgi by microtubules casts doubt on this notion.
Perhaps, instead, the disassembly process facilitates the allotment of the interphase Golgi ribbon into two populations, each organized directly or indirectly by the microtubules of a developing half-spindle Fig. In fact, the early segregation of Golgi membranes into two centrosome-associated populations see Figs. This mode of partitioning shares general features with the organization and separation of chromosomes during cell division, a paradigm that should provide a useful framework to facilitate the future dissection of the inheritance mechanism for the Golgi apparatus.
We would like to thank W. Balch for supplying the Sar1p plasmids; M. Lowe for assistance in purification of Sar proteins; H. Hauri, D. Mundy, M. Renz, and T. Suganuma for supplying antibodies; A. Stokes and P. Jordan for technical assistance with microscopy; and J. Herreros, M.
Lowe, and C. Ruhrberg for critical review of the manuscript. Transport inhibition by a dominant mutant of Sar1p mSar1p. In cells injected with mSar1p asterisks all markers analyzed accumulated in the ER. Arrowheads in b and c point to the nuclear envelope staining which is characteristic for ER localization. In the presence of mSar1p, exit from the ER was inhibited for the Golgi resident giantin. Analysis of mitotic Golgi fragmentation in mSar1p injected cells.
Projections of 10—25 z-sections 0. Direct visualization of Golgi membranes during mitosis. A Images were acquired every 5 min to see changes in Golgi morphology indicating that cells were in late G2 or the beginning of prophase. Thereafter images were acquired every minute, and cells were followed through all phases of mitosis. Images in a—d show early prophase cells before the breakdown of the nuclear envelope.
In e—h the prophase to metaphase transition is shown, and i shows the same cell in telophase. Arrows in a point to Golgi fragments organized around the position of the emerging spindle poles. B A series of images 1 min interval acquired at the prophase to metaphase transition are shown.
Mitotic Golgi fragments are designated by arrows. A quicktime movie comprising the images acquired will be available on the internet. Analysis of HeLa cell mitotic Golgi fragment dynamics and subcellular localization. A Images were acquired every 10 s to track Golgi fragment movements in the future polar regions during the prophase to metaphase transition.
Asterisks in 0 indicate Golgi fragments associated with the spindle poles identified by phase microscopy and subsequent location of metaphase chromosomes. Dotted lines indicate the emergence of a stable, non-mobile population of Golgi group 1 , and a population that reorganizes and disperses during the period of observation group 2.
Arrowheads indicate Golgi fragments stably associated with the spindle poles during the entire period of observation. B Dynamics of Golgi fragments in metaphase cells were analyzed by confocal microscopy acquiring five z-sections every 20 s. Shown is a two-dimensional projection of the z-series of images. Metaphase Golgi fragments are stable see arrowheads and undergo only insignificant redistribution.
Arrows designate membranes that appear to be distributing between the two separating centrosomes. Appearance of Golgi membranes and microtubules during mitosis in PtK1 cells. Exponentially growing PtK1 cells were fixed and stained for the Golgi marker GM green , a—g or mitochondria see Material and Methods; green , h and microtubules red.
Arrowheads b designate Golgi fragments that were frequently detected on either side of the nuclear envelope by the earliest indications of entrance into mitosis. Arrows indicate Golgi fragments in proximity to aster microtubules d , or concentrated around spindle poles g. Organization of mitotic Golgi membranes by microtubules in living PtK1 cells. Images were taken at s intervals. Still images highlight the concentration of mitotic Golgi arrowheads on either side of the metaphase plate arrows in first panel , presumably in association with elements of the mitotic spindle.
This peri-spindle distribution of mitotic Golgi was maintained while Golgi membranes were partitioned into the daughter cells. Two-dimensional projections of the z-sections are shown.
After disruption of the mitotic spindle with nocodazole, the peri-spindle organization of Golgi clusters redistributes d , with mitotic Golgi appearing more frequently at the cell periphery. Analysis of nocodazole induced Golgi fragmentation in mSar1p injected cells.
In e and f , cells were first treated with nocodazole for 2 h, microinjected and subsequently incubated in the presence of nocodazole for 1 h.
Microinjected cells marked by asterisks were identified by the coinjected BSA not shown. Microinjection of mSar1p results in the accumulation of ERGIC 53 in the ER but does not interfere with the formation and distribution of the Golgi fragments marked by arrowheads in b ; in noninjected cells ERGIC 53 and giantin often appear to colocalize in peripheral Golgi fragments marked by arrows.
Model for Golgi apparatus disassembly and partitioning during mitosis. The rounding process becomes detectable during late prophase, but the preparatory steps begin during G2, when the focal adhesions FA undergo selective disassembly. This process is essential to achieve the progressive retraction of the cell margins and the formation of the actomyosin cortex, which are necessary for the cell rounding in metaphase Cadart et al.
Cell rounding is necessary to create a symmetric cell organization that allows the kinetochore MTs to capture the chromosomes. In fact, a reduced rounding can cause the scattering of chromosomes, which decreases the probability of being captured by the kinetochore MTs.
Besides, an asymmetric cell geometry can induce spindle deformations, resulting in the splitting of the spindle poles and formation of acentriolar spindles, thus increasing the likelihood of multipolar cell division and aneuploidy.
Therefore, alterations of mitotic rounding can affect spindle morphology, chromosome segregation, and timely mitotic progression Champion et al. In addition, the geometry of the cell during mitosis also influences the orientation of the spindle and, as a consequence, the direction of the cell division axis, and thus tissue morphogenesis and differentiation Morin and Bellaiche, Collectively, these findings emphasize that correct cell geometry and the controlled redistribution of cell components are crucial for the daughter cells fate, and errors in the process can lead to the development of diseases Lancaster et al.
Entry into mitosis is also accompanied by an extensive reorganization of subcellular organelles, which undergo stereotyped reorganization of the structure and localization Champion et al. For instance, during G2 the centrosomes move from the perinuclear area to the center of the nucleus in order to be disengaged and separated Sutterlin and Colanzi, The reorganization of organelle morphology can range from the full disassembly of the GC to the subtle modifications of the endosomes Lowe and Barr, ; Jongsma et al.
In the next paragraphs, we will summarize the current knowledge about the mitotic fate of the intracellular organelles, with a focus on the GC, and referring to several recent reviews for more mechanistic details. The GC has a pivotal role in the secretory pathway, as it is involved in the modification and sorting of cargoes Wei and Seemann, In mammalian cells, the GC is characterized by a ribbon structure, which is composed of several polarized stacks of cisternae that are laterally connected by tubules Lowe, All these proteins act as membrane tethers and concur in the stacking of the cisternae and in directing the formation of the membranous tubules connecting the stacks Xiang and Wang, ; Witkos and Lowe, Moreover, Golgi unlinking requires the fission-inducing protein BARS to cleave the tubules connecting the stacks Hidalgo Carcedo et al.
For more mechanistic details the reader is referred to several reviews Lowe and Barr, ; Corda et al. During prophase Figure 2C , the activation of CDK1 leads to the phosphorylation of additional sites on golgins and GRASPs, resulting in complete inhibition of the membrane tethering processes.
Then, during telophase and cytokinesis, the dispersed Golgi proteins and membranes are gradually reassembled into a GC in each of the daughter cells Figure 2E ; Shorter and Warren, ; Altan-Bonnet et al. Figure 2.
Schematic representation of mitotic redistribution and inheritance of organelles and their connection with the cell cycle. In mammalian cells, during mitosis, the organelles are subjected to complex structural reorganizations. A,B During G2, the Golgi ribbon is converted into isolated stacks, and the centrosomes are separated. D During metaphase, several organelle-based protein machineries e. E After the formation of the cleavage furrow, the cells are ready to complete the cytokinesis process, which requires mitochondria fission and endosome traffic d.
Impairment of one of the membrane-based processes can cause several defects in correct completion of mitosis, with potential repercussions on tissue homeostasis and diseases development. Adapted with permission from Ayala and Colanzi Importantly, the disassembly of the GC is also a requirement for mitotic entry. It is already known that the structure and localization of the GC can be modulated by the centrosome through multiple mechanisms Sutterlin and Colanzi, ; Rios, The centrosome is composed of two centrioles enclosed by pericentriolar material PCM , which consists of a thick shell of multiprotein complexes.
The centrosome is positioned at the cell center, close to the nucleus. Following a polarization stimulus, it is reoriented in the direction of the leading edge of the cell Pouthas et al. The centrosomal MTs form radial fibers that guide the positioning of the Golgi membranes toward the cell center thanks to dynein, which is a minus end-directed motor complex Rios, that is recruited at the GC by Golgin Yadav et al.
Also, the actin cytoskeleton contributes to the maintenance of the ribbon, as it forms tracks for actin-based motors Valderrama et al. The GC-based MT nucleation is crucial not only for the structural integrity of the GC, but also for the formation of asymmetric MTs that are essential for the orientation of the GC toward the leading edge during migration Vinogradova et al. Yet, the significance of the GC-centrosome proximity and of the ribbon organization are not completely understood.
In particular, the knockdown of the golgin GMAP or Golgin induces the unlinking into separated stacks, which are still able to transport cargoes to the cell surface but that become unable to direct the secretion toward specific domains of the plasma membrane PM at the leading edge.
As a result, the directional persistence of cell migration is reduced Yadav et al. Additionally, experiments based on the expression of various N-terminal fragments of AKAP led to the conclusion that the proximity of the GC to the centrosome, but not the presence of an intact ribbon, is the crucial factor for optimal directional cell migration Hurtado et al.
However, experiments based on RPE1 cells in which GM was knocked out led to the conclusion that a close association of the GC with the centrosome is not required for cell migration or protein transport Tormanen et al. Therefore, more investigations are needed to better understand the functional consequences of perturbations of the GC structure, or of its proximity to the centrosome.
The structural reorganization of the GC during the cell cycle appears to be coordinated with those of the centrosome Sutterlin and Colanzi, The centrosomes are duplicated during S-phase; then, during G2, they are pulled apart, in coincidence with the severing of the Golgi ribbon Figure 2B ; Persico et al. Thus, the GC is segregated into two groups of stacks, each of which is localized in proximity to a separated centrosome Figure 2C.
Furthermore, during this phase, the membranes of the IC remain closely associated with the centrosomes and become detached from the bulk of the GC, suggesting that the IC maintains its identity during mitosis and provides an intermediate station for Golgi dispersal Marie et al. Defects in assembly and duplication of the centrosomes, and the consequent problems in MT nucleation, are the primary cause of the formation of aberrant spindles. In support of a functional GC-centrosome relationship, the G2-specific Golgi ribbon unlinking acts as a controller of the centrosomal recruitment of Aurora A Persico et al.
In particular, Barretta et al. Cells containing DAB polymers in the Golgi stacks entered into mitosis normally, but they arrested in metaphase with intact Golgi clusters associated with monopolar spindles, which caused SAC activation. Artificial disassembly of the GC relieved this block, suggesting that the disassembly of the Golgi stacks is required for progression through mitosis Guizzunti and Seemann, ; Wei and Seemann, In addition, several reports have shown a direct role of Golgi matrix proteins in assisting spindle formation.
For example, the N-terminal domain of GM includes a nuclear localization signal NLS that has been shown to be essential for proper spindle assembly Wei et al. During interphase, the NLS is masked by the interaction with the Golgi matrix protein p CDK1-mediated phosphorylation of GM dissociates p from GM, and this triggers a crucial pathway of mitotic disassembly of the Golgi stacks Nakamura et al.
Probably correlated to this function, depletion of GM causes the formation of over duplicated centrosomes and multipolar spindles during mitosis Table 1 , resulting in metaphase arrest and cell death Kodani and Sutterlin, To further support the functional connection of the GC with the spindle, several reports have shown evidence of Golgi-associated proteins that influence spindle formation and mitotic progression.
In this regard, the GM interactor p becomes associated with the mitotic spindle throughout mitosis. Strikingly, p depletion causes spindle abnormalities, chromosome defects, and cytokinesis failure Table 1 ; Radulescu et al.
The list of Golgi-associated proteins with roles in spindle formation is not limited to the GMbased protein complex. For instance, depletion of the Golgi-associated phosphoinositide phosphatase SAC1 causes perturbations of Golgi architecture and spindle abnormalities Table 1 ; Liu et al. In addition, tankyrase-1 is an ADP-ribosyltransferase that is associated with the Golgi in interphase, and relocates to the spindle poles during mitosis.
Its depletion causes mitotic arrest with abnormal chromosome segregation, bipolar spindle formation, and failure of telomere separation Table 1 ; Chang et al. Depletion of Miki induces a pseudometaphase state that leads to the formation of multinucleated cells Table 1 ; Ozaki et al. Another Golgi-associated protein implicated in cell cycle control is the Radinteracting protein RINT-1, whose depletion causes partial Golgi fragmentation, centrosome amplification during interphase, and increased formation of multiple spindle poles that culminate in frequent chromosome missegregation Table 1 ; Lin et al.
More in general, the spindle recruits and directs the inheritance of Golgi matrix proteins that are involved in the formation of the Golgi ribbon, while a minimal set of proteins and membranes sufficient to reassemble functional Golgi stacks are inherited independently of the spindle Wei and Seemann, It could be speculated that the Golgi matrix proteins recruited by the spindle are not simple passengers, but acquire different mitosis-specific functions.
In support of this possibility, during mitosis, the small GTPase Arf1 becomes inactive and dissociates from the Golgi membranes Altan-Bonnet et al. If Arf1 is artificially kept active, Golgi membranes do not fragment, and the peripheral proteins remain associated with the GC throughout mitosis. These cells enter mitosis, but exhibit gross defects in chromosome segregation and cytokinetic furrow formation, resulting in multinucleation Altan-Bonnet et al.
Thus, there is a substantial amount of evidence to conclude that an active functional interplay between the GC and the centrosome is crucial for spindle formation and, hence, for accurate segregation of the genetic material. The ER is a large continuous membranous organelle that is responsible for the synthesis of the majority of the integral membrane proteins and lipids. The ER constitutes a vast network of cisternae and tubules spread across the cytosol, and establishes contacts with several subcellular compartments Phillips and Voeltz, The ER undergoes marked structural modifications during mitosis.
Specifically, the cisternae are transformed into mixed populations of tubules, the extent of which varies among cell lines Puhka et al. In addition, during late prophase, the nuclear envelope is disassembled and its membranes reabsorbed into the bulk of the ER to expose the chromatin to the spindle apparatus.
In prometaphase, the ER is split into two large pools of membranes that maintain continuity throughout mitosis Lu et al. During anaphase and telophase, after chromosomal segregation, the nuclear envelope reassembles, and this marks the beginning of the reorganization of the ER compartment, although the underlying molecular mechanisms are poorly understood Schwarz and Blower, The ER is also a major hub for intracellular organization and signaling Jongsma et al.
Membrane CSs have crucial functions in inter-organelle signaling and lipid transfer. Considering its role as a major organizing compartment, it is likely that the inheritance of the ER is regulated by yet unknown control mechanisms. During mitosis, the density of these specific CSs is decreased. Also, the average distance between the PM and the closest ER in mitosis is increased. The membranous endocytic system mediates the traffic of lipids, proteins, and other molecules among various intracellular locations.
These features have an essential impact on signal transduction and nutrient acquisition. Even if the various cellular functions of the endolysosomal system are extensively investigated, the mechanisms of endosome inheritance are marginally known.
The current view is that endosomes and lysosomes remain intact during mitosis, and that during cytokinesis these organelles accumulate in the proximity of the MTOC Bergeland et al.
Despite this limited knowledge, an interesting aspect is that during prophase, MT-dependent motors induce the clustering of Rabpositive endosomes around the centrosomes Figures 2C,c. This localization is important to prevent their transport to the PM and to bring MT-nucleating proteins to the centrosome Hehnly and Doxsey, For this reason, Rab11 relocation to the centrosome is necessary for the formation of a functional and properly oriented mitotic spindle.
The clustered Rab11 endosomes are then segregated by the mitotic spindles between the daughter cells Figures 2D,E ; Hehnly and Doxsey, Moreover, in line with the proposed role of membrane reservoir in mitotic cells, during anaphase, the recycling endosomes are transported toward the cleavage furrow Figure 2E ; Bergeland et al.
The peroxisomes are organelles involved in fatty acid and energy metabolism. During prophase, they are associated with the MT network and remain clustered around the spindle poles Figures 2C,D,c. Throughout telophase, they are repositioned around the reforming nucleus of each daughter cell Figure 2E ; Hettema and Motley, Importantly, an intriguing connection of peroxisome inheritance with tissue development has been demonstrated.
The peroxisome-associated protein PEX11b has been found to be essential for the differentiation of the skin, which undergoes a continuous renewal that is required to ensure a healthy tissue turnover Asare et al. Specifically, the peroxisomes of PEX11b-depleted cells are functional, indicating a marginal role of this protein for the most classical peroxisome roles.
However, while in control cells the peroxisomes localize at the spindle poles during mitosis, in PEX11b knockdown cells they fail to localize properly, resulting in a mitotic delay and SAC activation Asare et al. Furthermore, PEX11b deficiency is associated to uncontrolled rotations of the spindle, which should normally be oriented perpendicularly to the basal membrane. The localization of peroxisomes at the spindle poles is the crucial factor, as their artificial relocalization to the cell cortex, or the spindle midzone, is sufficient to cause the alterations of spindle orientation.
Importantly, in mouse embryos that are knockdown of PEX11b, the inability of the basal stem cells to orient their spindle perpendicularly to the basal membrane also led to alterations of their differentiation.
These perturbations had severe effects, as the epidermis showed hyperproliferation and increased expression of terminal differentiation markers in basal cells, which is a feature typically associated with cancer Asare et al.
Mitochondria are the energy factories of cells and are characterized by the presence of a double membrane. As metaphase ends, each centromere divides in two, separating the two sister chromatids. During anaphase, the poles move apart as nonkinetochore microtubules from opposite poles slide past each other, lengthening the cell. Simultaneously, the spindle fibers contract, pulling sister chromatids away from each other toward opposite poles.
Tubulin subunits are removed close to the kinetochore ends of the microtubules. In telophase, a nuclear envelope forms around each set of chromosomes and the nucleoli reappear. Finally, the spindle apparatus is disassembled. During animal cytokinesis, a ring of actin microfilaments called the contractile ring constricts around the cell, like the tightening of an imaginary belt.
This produces a groove in the cell surface called the cleavage furrow approximately at the site of the old metaphase plate. The cleavage furrow deepens until the cell is literally pinched into two daughter cells, each with one nucleus containing an identical set of chromosomes.
The cell grows and accumulates the building blocks of chromosomal DNA and the associated proteins as well as sufficient energy reserves to complete the task of replicating each chromosome in the nucleus. The synthesis phase of interphase takes the longest because of the complexity of the genetic material being duplicated.
Throughout interphase, nuclear DNA remains in a semi-condensed chromatin configuration. In the S phase, DNA replication results in the formation of identical pairs of DNA molecules, sister chromatids, that are firmly attached to the centromeric region. The centrosome is duplicated during the S phase. The two centrosomes will give rise to the mitotic spindle, the apparatus that orchestrates the movement of chromosomes during mitosis.
At the center of each animal cell, the centrosomes of animal cells are associated with a pair of rod-like objects, the centrioles, which are at right angles to each other. Centrioles help organize cell division. Centrioles are not present in the centrosomes of other eukaryotic species, such as plants and most fungi. In the G 2 phase, the cell replenishes its energy stores and synthesizes proteins necessary for chromosome manipulation. Some cell organelles are duplicated, and the cytoskeleton is dismantled to provide resources for the mitotic phase.
There may be additional cell growth during G 2. The final preparations for the mitotic phase must be completed before the cell is able to enter the first stage of mitosis. During the multistep mitotic phase, the cell nucleus divides, and the cell components split into two identical daughter cells.
The mitotic phase is a multistep process during which the duplicated chromosomes are aligned, separated, and move into two new, identical daughter cells. The first portion of the mitotic phase is called karyokinesis or nuclear division. The second portion of the mitotic phase, called cytokinesis, is the physical separation of the cytoplasmic components into the two daughter cells. Karyokinesis, also known as mitosis, is divided into a series of phases prophase, prometaphase, metaphase, anaphase, and telophase that result in the division of the cell nucleus.
Stages of the Cell Cycle : Karyokinesis or mitosis is divided into five stages: prophase, prometaphase, metaphase, anaphase, and telophase.
The images at the bottom were taken by fluorescence microscopy hence, the black background of cells artificially stained by fluorescent dyes: blue fluorescence indicates DNA chromosomes and green fluorescence indicates microtubules spindle apparatus. The membranous organelles such as the Golgi apparatus and endoplasmic reticulum fragment and disperse toward the periphery of the cell.
The nucleolus disappears and the centrosomes begin to move to opposite poles of the cell. Microtubules that will eventually form the mitotic spindle extend between the centrosomes, pushing them farther apart as the microtubule fibers lengthen.
0コメント