The Golgi-associated Protein GRASP65 Regulates Spindle Dynamics and Is Essential for Cell Division

At the onset of mitosis, the pericentriolar Golgi apparatus of mammalian cells is converted into small fragments, which are dispersed throughout the cytosol. The Golgi-associated protein GRASP65 is involved in this process. To address the role of GRASP65 in mitotic Golgi fragmentation, we depleted the protein from HeLa cells by RNAi. In the absence of GRASP65, the number of cisternae per Golgi stack is reduced without affecting the overall organization of Golgi membranes and protein transport. GRASP65-depleted cells entered mitosis, but accumulated in metaphase with condensed chromatin and multiple aberrant spindles and eventually died. Although Centrin2 and g-tubulin were detected in two of the spindle poles, the other spindle poles contained g-tubulin, but not Centrin2. Furthermore, we provide evidence that the expression of the C-terminus of GRASP65 interferes with entry of cells into mitosis. Our results suggest the requirement for GRASP65 in the regulation of spindle dynamics rather than a direct role in the stacking of Golgi cisternae. This novel function is in addition to the previously established negative role of GRASP65 at the G2/M transition, which is mediated by its C-terminus.

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Melatonin reduces oxidative damage in mouse granulosa cells via restraining JNK-dependent autophagy

Reproduction ◽

10.1530/rep-18-0002 ◽

2018 ◽

Vol 155 (3) ◽

pp. 307-319 ◽

Cited By ~ 7

Author(s):

Yan Cao ◽

Ming Shen ◽

Yi Jiang ◽

Shao-chen Sun ◽

Honglin Liu

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Mammalian Cells ◽

Therapeutic Strategies ◽

Oxygen Species ◽

Protective Effects ◽

Follicular Atresia ◽

Direct Role ◽

Jnk Pathway

Oxidative stress-induced granulosa cell (GCs) injury is believed to be a common trigger for follicular atresia. Emerging evidence indicates that excessive autophagy occurs in mammalian cells with oxidative damage. N-acetyl-5-methoxytrypamine (melatonin) has been shown to prevent GCs from oxidative injury, although the exact mechanism remains to be elucidated. Here, we first demonstrated that the suppression of autophagy through the JNK/BCL-2/BECN1 signaling is engaged in melatonin-mediated GCs protection against oxidative damage. Melatonin inhibited the loss of GCs viability, formation of GFP-MAP1LC3B puncta, accumulation of MAP1LC3B-II blots, degradation of SQSTM1 and the expression of BECN1, which was correlated with impaired activation of JNK during oxidative stress. On the other hand, blocking of autophagy and/or JNK also reduced the level of H2O2-induced GCs death, but failed to further restore GCs viability in the presence of melatonin. Particularly, the suppression of autophagy provided no additional protective effects when GCs were pretreated with JNK inhibitor and/or melatonin. Importantly, we found that the enhanced interaction between BCL-2 and BECN1 might be a responsive mechanism for autophagy suppression via the melatonin/JNK pathway. Moreover, blocking the downstream antioxidant system of melatonin using specific inhibitors further confirmed a direct role of melatonin/JNK/autophagy axis in preserving GCs survival without scavenging reactive oxygen species (ROS). Taken together, our findings uncover a novel function of melatonin in preventing GCs from oxidative damage by targeting JNK-mediated autophagy, which might contribute to develop therapeutic strategies for patients with ovulation failure-related disorders.

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The multiple facets of the Golgi reassembly stacking proteins

Biochemical Journal ◽

10.1042/bj20101540 ◽

2011 ◽

Vol 433 (3) ◽

pp. 423-433 ◽

Cited By ~ 64

Author(s):

Fabian P. Vinke ◽

Adam G. Grieve ◽

Catherine Rabouille

Keyword(s):

Mammalian Cells ◽

Secretory Pathway ◽

Protein Transport ◽

Mitotic Checkpoint ◽

Biochemical Properties ◽

C Terminus ◽

Unconventional Secretion ◽

Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

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K-fiber bundles in the mitotic spindle are mechanically reinforced by Kif15

Molecular Biology of the Cell ◽

10.1091/mbc.e20-06-0426 ◽

2021 ◽

Author(s):

Marcus A Begley ◽

April L Solon ◽

Elizabeth Mae Davis ◽

Michael Grant Sherrill ◽

Ryoma Ohi ◽

...

Keyword(s):

Chromosome Segregation ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Binding Ability ◽

Microtubule Binding ◽

Mechanical Integrity ◽

Spindle Poles ◽

Rigor State ◽

Physical Response

The mitotic spindle, a self-constructed microtubule-based machine, segregates chromosomes during cell division. In mammalian cells, microtubule bundles called kinetochore-fibers (k-fibers) connect chromosomes to the spindle poles. Chromosome segregation thus depends on the mechanical integrity of k-fibers. Here, we investigate the physical and molecular basis of k-fiber bundle cohesion. We detach k-fibers from poles by laser ablation-based cutting, thus revealing the contribution of pole-localized forces to k-fiber cohesion. We then measure the physical response of the remaining kinetochore-bound segments of the k-fibers. We observe that microtubules within ablated k-fibers often splay apart from their minus-ends. Furthermore, we find that minus-end clustering forces induced by ablation seem at least partially responsible for k-fiber splaying. We also investigate the role of the k-fiber-binding kinesin-12 Kif15. We find that pharmacological inhibition of Kif15-microtubule binding reduces the mechanical integrity of k-fibers. In contrast, inhibition of its motor activity but not its microtubule binding ability, i.e., locking Kif15 into a rigor state, does not greatly affect splaying. Altogether, the data suggest that forces holding k-fibers together are of similar magnitude to other spindle forces, and that Kif15, acting as a microtubule crosslinker, helps fortify and repair k-fibers. This feature of Kif15 may help support robust k-fiber function and prevent chromosome segregation errors. [Media: see text] [Media: see text] [Media: see text]

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Gαq Regulates the Development of Rheumatoid Arthritis by Modulating Th1 Differentiation

Mediators of Inflammation ◽

10.1155/2017/4639081 ◽

2017 ◽

Vol 2017 ◽

pp. 1-9 ◽

Cited By ~ 7

Author(s):

Dashan Wang ◽

Yuan Liu ◽

Yan Li ◽

Yan He ◽

Jiyun Zhang ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Bone Marrow ◽

Immune Regulation ◽

Inflammatory Arthritis ◽

Mammalian Cells ◽

Th1 Cells ◽

Th1 Cell ◽

Th1 Cytokine ◽

Negative Role

The Gαq-containing G protein, an important member ofGq/11class, is ubiquitously expressed in mammalian cells. Gαq has been found to play an important role in immune regulation and development of autoimmune disease such as rheumatoid arthritis (RA). However, how Gαq participates in the pathogenesis of RA is still not fully understood. In the present study, we aimed to find out whether Gαq controls RA via regulation of Th1 differentiation. We observed that the expression of Gαq was negatively correlated with the expression of signature Th1 cytokine (IFN-γ) in RA patients, which suggests a negative role of Gαq in differentiation of Th1 cells. By using Gαq knockout (Gnaq−/−) mice, we demonstrated that loss of Gαq led to enhanced Th1 cell differentiation. Gαq negative regulated the differentiation of Th1 cell by modulating the expression of T-bet and the activity of STAT4. Furthermore, we detected the increased ratio of Th1 cells inGnaq−/−bone marrow (BM) chimeras spontaneously developing inflammatory arthritis. In conclusion, results presented in the study demonstrate that loss of Gαq promotes the differentiation of Th1 cells and contributes to the pathogenesis of RA.

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The Role of the Centrosome in Development and Progression of Breast Cancer

Microscopy and Microanalysis ◽

10.1017/s1431927600028981 ◽

2001 ◽

Vol 7 (S2) ◽

pp. 582-583

Author(s):

W. Lingle ◽

J. Salisbury ◽

S. Barrett ◽

V. Negron ◽

C. Whitehead

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Microtubule Organizing Center ◽

Daughter Cells ◽

Spindle Poles ◽

Sister Chromatids ◽

Close Proximity ◽

Interphase Cells ◽

Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

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Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle

Journal of Cell Science ◽

10.1242/jcs.111.17.2551 ◽

1998 ◽

Vol 111 (17) ◽

pp. 2551-2561 ◽

Cited By ~ 2

Author(s):

C.M. Whitehead ◽

J.B. Rattner

Keyword(s):

Cell Division ◽

Microtubule Bundle ◽

Spindle Poles ◽

Centrosome Separation ◽

Spindle Dynamics ◽

Spindle Microtubules ◽

Protein Components ◽

The Stability

The BimC family of kinesin like proteins are involved in spindle dynamics in a wide variety of organisms. The human member of this family, HsEg5, has been implicated in centrosome separation during prophase/prometaphase and in the organization of in vitro mitotic asters. HsEg5 displays a complex distribution during mitosis, associating with the centrosomes, spindle microtubules, specific regions of the intracellular bridge and a microtubule bundle that forms in association with the post-mitotic migration of the centrosome. In an effort to determine the function of HsEg5 during late mitotic events and refine its proposed function during early mitotic centrosome separation, we microinjected antibodies specific to HsEg5 into HeLa cells during various stages of mitosis. In the presence of HsEg5 antibodies we find that the microtubule arrays responsible for both pre- and post-mitotic centrosome movement never form. Similarly, the microtubule bundle within the intracellular bridge becomes prematurely altered following karyokinesis resulting in the loss of the microtubule array at either end of the bridge. In addition, some peri-centrosomal material at the spindle poles becomes fragmented and the distribution of the spindle protein NuMA becomes more concentrated at the minus ends of the spindle microtubules. Our study also provides direct evidence that there is a link between post-mitotic centrosome migration and Golgi complex positioning and reformation following mitosis. We conclude that HsEg5 plays a recurrent role in establishing and/or determining the stability of specific microtubule arrays that form during cell division and that this role may encompass the ability of HsEg5 to influence the distribution of other protein components associated with cell division

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CRM1-Dependent Function of a cis-Acting RNA Export Element

Molecular and Cellular Biology ◽

10.1128/mcb.22.7.2057-2067.2002 ◽

2002 ◽

Vol 22 (7) ◽

pp. 2057-2067 ◽

Cited By ~ 58

Author(s):

Ileana Popa ◽

Matthew E. Harris ◽

John E. Donello ◽

Thomas J. Hope

Keyword(s):

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Hepatitis Virus ◽

Regulatory Element ◽

Viral Proteins ◽

Direct Role ◽

Independent Manner ◽

Export Pathway ◽

C Terminus

ABSTRACT Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.

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Nudel Modulates Kinetochore Association and Function of Cytoplasmic Dynein in M Phase

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0345 ◽

2007 ◽

Vol 18 (7) ◽

pp. 2656-2666 ◽

Cited By ~ 57

Author(s):

Yun Liang ◽

Wei Yu ◽

Yan Li ◽

Lihou Yu ◽

Qiangge Zhang ◽

...

Keyword(s):

Protein Transport ◽

Cytoplasmic Dynein ◽

Mitotic Progression ◽

Kinetochore Protein ◽

Nuclear Envelope Breakdown ◽

Spindle Poles ◽

M Phase ◽

And Function ◽

Force Generator

The microtubule-based motor cytoplasmic dynein/dynactin is a force generator at the kinetochore. It also transports proteins away from kinetochores to spindle poles. Regulation of such diverse functions, however, is poorly understood. We have previously shown that Nudel is critical for dynein-mediated protein transport, whereas mitosin, a kinetochore protein that binds Nudel, is involved in retention of kinetochore dynein/dynactin against microtubule-dependent stripping. Here we demonstrate that Nudel is required for robust localization of dynein/dynactin at the kinetochore. It localizes to kinetochores after nuclear envelope breakdown, depending mostly (∼78%) on mitosin and slightly on dynein/dynactin. Depletion of Nudel by RNA interference (RNAi) or overexpression of its mutant incapable of binding either Lis1 or dynein heavy chain abolishes the kinetochore protein transport and mitotic progression. Similar to mitosin RNAi, Nudel RNAi also leads to increased stripping of kinetochore dynein/dynactin in the presence of microtubules. Taking together, our results suggest a dual role of kinetochore Nudel: it activates dynein-mediated protein transport and, when interacting with both mitosin and dynein, stabilizes kinetochore dynein/dynactin against microtubule-dependent stripping to facilitate the force generation function of the motor.

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The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties

Journal of Cell Science ◽

10.1242/jcs.107.4.1019 ◽

1994 ◽

Vol 107 (4) ◽

pp. 1019-1029 ◽

Cited By ~ 12

Author(s):

H. Hennekes ◽

E.A. Nigg

Keyword(s):

Mammalian Cells ◽

Membrane Binding ◽

Proteolytic Cleavage ◽

Single Point Mutation ◽

Lamin A ◽

Binding Properties ◽

Nuclear Lamins ◽

C Terminus ◽

Membrane Attachment

Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.

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Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

10.1101/2021.09.29.462463 ◽

2021 ◽

Author(s):

Alessandro Dema ◽

Jeffrey van Haren ◽

Torsten Wittmann

Keyword(s):

Mitotic Spindle ◽

Mammalian Cells ◽

Live Cells ◽

Cell Cortex ◽

Reversible Inactivation ◽

Molecular Systems ◽

Spindle Poles ◽

Spindle Dynamics ◽

Spindle Function ◽

Transient Relaxation

Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

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