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BioRxiv : the Preprint Server For... Apr 2024Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from...
Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.
PubMed: 38659898
DOI: 10.1101/2024.04.12.589246 -
BioRxiv : the Preprint Server For... Apr 2024Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms...
Microtubule-based spindle formation is essential to faithful chromosome segregation during cell division. In many animal species, the oocyte meiotic spindle forms without centrosomes, unlike most mitotic cells. Even in mitotic cells, centrosomes are sometimes dispensable for bipolar spindle formation. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly was normal after auxin-induced degradation of Ran-GEF but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown as well as in the spindle region during metaphase I and metaphase II. Through light and electron microscopy we found that the concentration of soluble tubulin in the metaphase II spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules from the meiotic spindle. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes. We present data supporting a model in which cytoplasmic organelles exclude cytoplasmic volume to drive concentration of tubulin within the nuclear/spindle envelope.
PubMed: 38659754
DOI: 10.1101/2024.04.19.590357 -
Development (Cambridge, England) May 2024The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning...
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Topics: Animals; Metaphase; Stress, Mechanical; Epithelial Cells; Spindle Apparatus; Drosophila melanogaster; Cell Division; Cell Polarity; Body Patterning; Myosin Type II; Embryo, Nonmammalian; Drosophila Proteins; Gastrulation
PubMed: 38639390
DOI: 10.1242/dev.202862 -
Cellular and Molecular Life Sciences :... Apr 2024Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule...
Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.
Topics: Animals; Mice; Biological Transport; Kinesins; Meiosis; Metaphase; Oocytes
PubMed: 38587639
DOI: 10.1007/s00018-024-05213-3 -
Frontiers in Endocrinology 2024Progesterone-primed cycles effectively suppress the pituitary LH surge during ovarian stimulation in oocyte donors and in the infertile population. Particularly in...
Micronized natural progesterone (Seidigestan) vs GnRH antagonists for preventing the LH surge during controlled ovarian stimulation (PRO_NAT study): study protocol of a randomized clinical trial.
INTRODUCTION
Progesterone-primed cycles effectively suppress the pituitary LH surge during ovarian stimulation in oocyte donors and in the infertile population. Particularly in oocyte donors, the use of synthetic progesterone (progestins) has been explored in prospective clinical trials, showing mixed results. This trial was designed to determine whether the use of micronized natural progesterone is as effective as the GnRH-antagonist protocol in terms of the number of mature oocytes (MII) retrieved in oocyte donation cycles as a primary outcome, and it also aims to explore the corresponding results in recipients as a secondary outcome.
METHODS
We propose a prospective, open-label, non-inferiority clinical trial to compare a novel approach for oocyte donors with a control group, which follows the standard ovarian stimulation protocol used in our institution. A total of 150 donors (75 in each group) will be recruited and randomized using a computer algorithm. After obtaining informed consent, participants will be randomly assigned to one of two ovarian stimulation protocols: either the standard GnRH antagonist or the oral micronized natural progesterone protocol. Both groups will receive recombinant gonadotropins tailored to their antral follicle count and prior donation experiences, if any. The primary outcome is the number of mature metaphase II (MII) oocytes. Secondary measures include treatment duration, pregnancy outcomes in recipients, as well as the economic cost per MII oocyte obtained in each treatment regimen. Analyses for the primary outcome will be conducted in both the intention-to-treat (ITT) and per-protocol (PP) populations. Each donor can participate only once during the recruitment period. The estimated duration of the study is six months for the primary outcome and 15 months for the secondary outcomes.
DISCUSSION
The outcomes of this trial have the potential to inform evidence-based adjustments in the management of ovarian stimulation protocols for oocyte donors.
CLINICAL TRIAL REGISTRATION
ClinicalTrials.gov, identifier, NCT05954962.
Topics: Female; Humans; Pregnancy; Gonadotropin-Releasing Hormone; Hormone Antagonists; Ovulation Induction; Progesterone; Progestins; Prospective Studies; Randomized Controlled Trials as Topic
PubMed: 38577571
DOI: 10.3389/fendo.2024.1350154 -
MBio May 2024Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin...
UNLABELLED
Condensin I is a pentameric complex that regulates the mitotic chromosome assembly in eukaryotes. The kleisin subunit CAP-H of the condensin I complex acts as a linchpin to maintain the structural integrity and loading of this complex on mitotic chromosomes. This complex is present in all eukaryotes and has recently been identified in spp. However, how this complex is assembled and whether the kleisin subunit is critical for this complex in these parasites are yet to be explored. To examine the role of PfCAP-H during cell division within erythrocytes, we generated an inducible PfCAP-H knockout parasite. We find that PfCAP-H is dynamically expressed during mitosis with the peak expression at the metaphase plate. PfCAP-H interacts with PfCAP-G and is a non-SMC member of the condensin I complex. Notably, the absence of PfCAP-H does not alter the expression of PfCAP-G but affects its localization at the mitotic chromosomes. While mitotic spindle assembly is intact in PfCAP-H-deficient parasites, duplicated centrosomes remain clustered over the mass of unsegmented nuclei with failed karyokinesis. This failure leads to the formation of an abnormal nuclear mass, while cytokinesis occurs normally. Altogether, our data suggest that PfCAP-H plays a crucial role in maintaining the structural integrity of the condensin I complex on the mitotic chromosomes and is essential for the asexual development of malarial parasites.
IMPORTANCE
Mitosis is a fundamental process for parasites, which plays a vital role in their survival within two distinct hosts-human and mosquitoes. Despite its great significance, our comprehension of mitosis and its regulation remains limited. In eukaryotes, mitosis is regulated by one of the pivotal complexes known as condensin complexes. The condensin complexes are responsible for chromosome condensation, ensuring the faithful distribution of genetic material to daughter cells. While condensin complexes have recently been identified in spp., our understanding of how this complex is assembled and its precise functions during the blood stage development of remains largely unexplored. In this study, we investigate the role of a central protein, PfCAP-H, during the blood stage development of . Our findings reveal that PfCAP-H is essential and plays a pivotal role in upholding the structure of condensin I and facilitating karyokinesis.
Topics: Humans; Adenosine Triphosphatases; DNA-Binding Proteins; Erythrocytes; Gene Knockout Techniques; Mitosis; Multiprotein Complexes; Plasmodium falciparum; Protozoan Proteins; Cell Nucleus Division
PubMed: 38564676
DOI: 10.1128/mbio.02850-23 -
ELife Apr 2024The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including...
The chromosomal passenger complex (CPC) is an important regulator of cell division, which shows dynamic subcellular localization throughout mitosis, including kinetochores and the spindle midzone. In traditional model eukaryotes such as yeasts and humans, the CPC consists of the catalytic subunit Aurora B kinase, its activator INCENP, and the localization module proteins Borealin and Survivin. Intriguingly, Aurora B and INCENP as well as their localization pattern are conserved in kinetoplastids, an evolutionarily divergent group of eukaryotes that possess unique kinetochore proteins and lack homologs of Borealin or Survivin. It is not understood how the kinetoplastid CPC assembles nor how it is targeted to its subcellular destinations during the cell cycle. Here, we identify two orphan kinesins, KIN-A and KIN-B, as bona fide CPC proteins in , the kinetoplastid parasite that causes African sleeping sickness. KIN-A and KIN-B form a scaffold for the assembly of the remaining CPC subunits. We show that the C-terminal unstructured tail of KIN-A interacts with the KKT8 complex at kinetochores, while its N-terminal motor domain promotes CPC translocation to spindle microtubules. Thus, the KIN-A:KIN-B complex constitutes a unique 'two-in-one' CPC localization module, which directs the CPC to kinetochores from S phase until metaphase and to the central spindle in anaphase. Our findings highlight the evolutionary diversity of CPC proteins and raise the possibility that kinesins may have served as the original transport vehicles for Aurora kinases in early eukaryotes.
Topics: Humans; Kinesins; Survivin; Trypanosoma; Cytoskeleton; Mitosis
PubMed: 38564240
DOI: 10.7554/eLife.93522 -
Animal Reproduction 2024The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the...
The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of transcripts in somatic tissues. Notably, the expression levels of in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo.
PubMed: 38562607
DOI: 10.1590/1984-3143-AR2023-0146 -
Cureus Mar 2024This case study presents a couple's journey through assisted reproductive technology (ART) experiencing two failed in vitro fertilization cycles. The couple underwent a...
This case study presents a couple's journey through assisted reproductive technology (ART) experiencing two failed in vitro fertilization cycles. The couple underwent a comprehensive examination, revealing the normal parameters for the female, but asthenoteratozoospermia in the male indicating high morphological defects and reduced sperm motility. Subsequently, intracytoplasmic sperm injection (ICSI) was planned. Despite retrieving six oocytes during ovum pickup (OPU), all blastocysts stopped growth on the second day, prompting a sperm chromatin test disclosing highly DNA-fragmented sperm. Platelet-rich plasma (PRP) therapy was initiated to improve sperm quality, along with frozen embryo transfer (FET). Sperm were incubated with PRP, yielding improved sperm motility and reduced sperm DNA fragmentation. OPU yielded five good-quality metaphase II (MII) oocytes, which were successfully fertilized with PRP-treated sperm, resulting in the formation of four blastocysts. These blastocysts were frozen and later used for FET, resulting in a positive pregnancy outcome and successful conception. This case highlights the importance of personalized intervention in addressing the infertility factor in males and achieving successful ART outcomes.
PubMed: 38562347
DOI: 10.7759/cureus.55378 -
BioRxiv : the Preprint Server For... Mar 2024The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein...
The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that SPC105R's C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for two activities that are critical for accurate chromosome segregation in meiosis I, lateral microtubule attachments and bi-orientation of homologs.
PubMed: 38559067
DOI: 10.1101/2024.03.14.585003