Matriptase drives dissemination of ovarian cancer spheroids by a PAR-2/PI3K/Akt/MMP9 signaling axis.

The transmembrane serine protease matriptase is a key regulator of both barrier-disruptive and protective epithelial cell-cell interactions. Elevated matriptase is a consistent feature of epithelial ovarian cancers (OvCa), where multicellular spheroids shed from the primary tumor into the peritoneal cavity are critical drivers of metastasis. Dynamic cell-to-cell adhesive contacts are required for spheroid formation and maintenance. Here, we show that overactive matriptase, reflected in an increased ratio of matriptase to its inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), disrupts cell-cell contacts to produce loose prometastatic spheroids that display increased mesothelial cell adhesion and submesothelial invasion. We show that these activities are dependent on the matriptase activation of a protease-activated receptor-2 (PAR-2) signaling pathway involving PI3K/Akt and MMP9-induced disruption of cell-cell adhesion by the release of the soluble E-cadherin ectodomain. These data reveal a novel pathological connection between matriptase activation of PAR-2 and disruption of cell-cell adhesion, and support the clinical investigation of this signaling axis as a therapeutic strategy for aggressive metastatic OvCa.

Intersection of Coagulation and Fibrinolysis by the Glycosylphosphatidylinositol (GPI)-Anchored Serine Protease Testisin.

Hemostasis is a delicate balance between coagulation and fibrinolysis that regulates the formation and removal of fibrin, respectively. Positive and negative feedback loops and crosstalk between coagulation and fibrinolytic serine proteases maintain the hemostatic balance to prevent both excessive bleeding and thrombosis. Here, we identify a novel role for the glycosylphosphatidylinositol (GPI)-anchored serine protease testisin in the regulation of pericellular hemostasis. Using in vitro cell-based fibrin generation assays, we found that the expression of catalytically active testisin on the cell surface accelerates thrombin-dependent fibrin polymerization, and intriguingly, that it subsequently promotes accelerated fibrinolysis. We find that the testisin-dependent fibrin formation is inhibited by rivaroxaban, a specific inhibitor of the central prothrombin-activating serine protease factor Xa (FXa), demonstrating that cell-surface testisin acts upstream of factor X (FX) to promote fibrin formation at the cell surface. Unexpectedly, testisin was also found to accelerate fibrinolysis by stimulating the plasmin-dependent degradation of fibrin and enhancing plasmin-dependent cell invasion through polymerized fibrin. Testisin was not a direct activator of plasminogen, but it is able to induce zymogen cleavage and the activation of pro-urokinase plasminogen activator (pro-uPA), which converts plasminogen to plasmin. These data identify a new proteolytic component that can regulate pericellular hemostatic cascades at the cell surface, which has implications for angiogenesis, cancer biology, and male fertility.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study.

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Study of Clinical Spectrum of Severe COVID-19 Infection in Elderly Patients and its Outcome-A Major Mumbai Tertiary Care Hospital Observations.

BACKGROUND: At 140 million, India has the second largest population of old people in the world, as per the 2011 census.1 The covid 19 pandemic has wreaked havoc in millions of lives. Elderly are especially vulnerable to COVID-19 and experience high morbidity and mortality as a result of immunosenescence. Age is independently linked with mortality, but age alone does not adequately capture the robustness of older adults who are a heterogeneous group. The current research was done in a tertiary healthcare hospital in Maharashtra to understand the clinical profile and factors that affected the outcome of elderly during the second wave of the COVID pandemic. METHOD: This was a single centre retrospective observational study done in a tertiary hospital which was admitting both covid and non-covid patients during the time of this study. All elderly patients admitted with COVID 19 disease in Covid ward and covid ICU (Intensive care unit) were included in the study. Their Demographic details, duration of illness, vital parameters, oxygen saturation, partial pressure of arterial oxygen compared to fraction of inspired oxygen (PaO2-FiO2 ratio) were recorded and also relevant investigations such as complete blood count, kidney function tests, liver function tests, arterial blood gases, chest X-rayand ECG (Electrocardiogram),CT scan of the brain, CSF(cerebrospinal fluid) studies and other tests where relevant were recorded. Inflammatory markers such as C-Reactive Protein (CRP), Ferritin, D-Dimer and Chest CT scan were noted. Clinical profiles and outcomes were noted till discharge or death. RESULTS: Among 231 patients that were included in this study, 81(35%) were female and 150 (65%) were male. Ninety-two patients died (39.8%) while 139 patients (60.2%) survived in our study. Majority of our patients (211;91.3%) presented in category E(pneumonia with respiratory failure) or category F(pneumonia with respiratory failure and multiorgan dysfunction syndrome). Factors which had a major impact on mortality were- a low PaO2-FiO2 ratio on admission, high C-Reactive Protein (CRP) levels, high d-dimer levels, a finding of bilateral ground glass opacities on x-ray, and need for invasive ventilation on admission. CONCLUSIONS: Elderly remain vulnerable to severe consequences of COVID-19 infection owing to the increasing comorbidities and immunosenescence in them. Prolonged oxygen therapy and intensive respiratory rehabilitation are the mainstays of effective management. Given the constant threat of mutating virus, masking, maintaining hand sanitization, vaccination and also caring for our elders while still maintaining social distance are our best bet against a fatal third wave.

Electric field-driven conformational changes in the elastin protein.

The formation of aggregates and amyloids, a hallmark of many protein misfolding diseases, depends on many intrinsic and extrinsic factors. Many approaches (in vitro, in vivo, and in silico) have been attempted to inhibit the aggregation process so that the progression of these diseases can be controlled. We investigate the effect of a static electric field (EF; 120 V cm-1 and 200 V cm-1) on the conformational change of elastin protein using light scattering, spectroscopy, and microscopy techniques. Laser light scattering and photoluminescence spectroscopy show the formation of fibrils of unexposed elastin with aging, whereas disruption of fibril formation with EF exposed elastin. The size of EF exposed elastin first increases and exhibits an apex, and subsequently decreases with an increasing time of exposure. We observed that a decrease in the size of EF exposed elastin depends on the strength of the EF, faster decrement at higher EF. FTIR data show that EF modifies elastin protein's secondary structures; it facilitates the interconversion of ß-sheets and turns into α-helix structures. The SEM images of unexposed and EF exposed elastin confirms the observation through light scattering and PL techniques. The effect of an EF on protein conformation and amyloids is promising to treat Parkinson's disease, a protein misfolding disease.

Interactive patches over amyloid-ß oligomers mediate fractal self-assembly.

The monomeric units of intrinsically disordered proteins self-assemble into oligomers, protofilaments, and eventually fibrils which may turn into amyloid. The aggregation of these proteins is primarily studied in bulk with no restriction on their degrees of freedom. Herein we experimentally demonstrate that amyloid-ß (Aß) aggregation under diffusion-limited conditions leads to its fractal self-assembly. Confocal microscopy and scanning electron microscopy with energy dispersion x-ray analysis were used to confirm that the fractal self-assemblies were formed from Aß rather than the salt present in the two supporting media: deionized water and phosphate buffered saline. The results from the molecular docking experiments implicated that electrostatic and hydrophobic patches on the solvent-accessible surface area of the Aß oligomers mediate the fractal self-assembly. These implications were tested with laser light scattering experiments on the oligomers formed by breaking mature fibrils of Aß through sonication, which were observed to self-assemble into fractals when sonicated solutions were drop casted. The electrostatic interactions modulate the fractal morphologies with pH of the solution, which leads to a morphological phase transition observed through the variation in their fractal dimension. These transitions provide experimental evidence for the existing theoretical framework in terms of different kinetic models. The higher surface-to-volume ratio of these fractal self-assemblies may have applications in drug delivery, biosensing, and other biomedical applications.

Fractal self-assembly and aggregation of human amylin.

Human amylin is an intrinsically disordered protein believed to have a central role in Type-II diabetes mellitus (T2DM). The formation of intermediate oligomers is a seminal event in the eventual self-assembled fibril structures of amylin. However, the recent experimental investigations have shown the presence of different self-assembled (oligomers, protofilaments, and fibrils) and aggregated structures (amorphous aggregates) of amylin formed during its aggregation. Here, we show that amylin under diffusion-limited conditions leads to fractal self-assembly. The pH and solvent sensitive fractal self-assemblies of amylin were observed using an optical microscope. Confocal microscopy and scanning electron microscopy (SEM) with energy dispersion X-ray analysis (EDAX) were used to confirm the fractal self-assembly of amylin in water and PBS buffer, respectively. The fractal characteristics of the self-assemblies and the aggregates formed during the aggregation of amylin under different pH conditions were investigated using laser light scattering. The hydropathy and the docking study indicated the interactions between the anisotropically distributed hydrophobic residues and polar/ionic residues on the solvent-accessible surface of the protein as the crucial interaction hot-spots for driving the self-assembly and aggregation of human amylin. The simultaneous presence of various self-assemblies of human amylin was observed through different microscopy techniques. The present study may help in designing different fractal-like nanomaterials with potential applications in drug delivery, sensing, and tissue engineering.

Glucose-induced structural changes and anomalous diffusion of elastin.

Elastin is the principal protein component of elastic fiber, which renders essential elasticity to connective tissues and organs. Here, we adopted a multi-technique approach to study the transport, viscoelastic, and structural properties of elastin exposed to various glucose concentrations (X=[gluc]/[elastin]). Laser light scattering experiments revealed an anomalous behavior (anomaly exponent, ß <0.6) of elastin. In this regime (ß <0.6), the diffusion constant decreases by 40% in the presence of glucose (X> 10), which suggests the structural change in elastin. We have observed a peculiar inverse temperature transition of elastin protein, which is a measure of structural change, at 40 °C through rheology experiments. Moreover, we observe its shift towards lower temperature with a higher X. FTIR revealed that the presence of glucose (X < 10) favors the formation of ß-sheet structure in elastin. However, for X > 10, dominative crowding effect reduces the mobility of protein and favors the increase in ß-turns and γ-turns by 25 ± 1% over the ß-sheet (ß-sheet decreases by 12 ± 0.8%) and α-helix (α-helix decreases by 13 ± 0.8%). The stiffness of protein is estimated through Flory characteristic ratio, C∞ and found to be increasing with X. These glucose-based structural changes in the elastin may explain the role of glucose in age-related issues of the skin.

Curcumin Complexed with Graphene Derivative for Breast Cancer Therapy.

In recent years, graphene-based materials complexed with drugs have been developed for application in cancer therapy, aimed at gaining synergistic effect. Here, we have prepared graphene oxide (GO) and graphene quantum dots (GQDs) with curcumin (Cur) in three different ratios (1:1, 1:3, and 1:5 w/v). We showed a successful complexation of GO and GQDs with Cur through various spectroscopy and microscopy techniques. The optical density of the complex through UV-vis spectroscopy showed less than 10% (for GQDs-Cur) and less than 20% (for GO-Cur) aggregation in 48 h, which confirms the stability of the complex. The UV-vis result estimates the loading efficiency of Cur to be 80 ± 1 and 83 ± 1% for GO-Cur and GQDs-Cur respectively. We tested the complexes GO-Cur and GQDs-Cur in different ratios as an anticancer drug against human breast cancer cell lines MCF-7 and MDA-MB-468 through the MTT assay. Following 48 h of incubation with the cell lines, a cell viability of more than 75% was observed in the case of GQDs & GO, while it was 40% in the case of Cur at a concentration of 100 µg/mL. The 1:1, 1:3, and 1:5 ratios of complexes enforced cell death ∼60, ∼80, and ∼95% at 100 µg/mL after 48 h of treatment, respectively. The optical images of cancerous cells treated with GO, GQDs, Cur, GO-Cur, and GQDs-Cur, at three different time intervals (0, 24, and 48 h), corroborated well with the results from the MTT assay in terms of the percentage of dead cells. The fluorescence images show a successful delivery of Cur drug inside the cancerous cell. The possible mechanism of killing of the cancerous cell with the complexes GO-Cur and GQDs-Cur is discussed. Moreover, this study opens a window to determine the mechanism of killing the cancerous cell.

Correction to: PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

The affiliation of Erik W. Martin is corrected in this paper.

Fibrinolysis and Inflammation in Venous Thrombus Resolution.

Clinical observations and accumulating laboratory evidence support a complex interplay between coagulation, inflammation, innate immunity and fibrinolysis in venous thromboembolism (VTE). VTE, which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), and the subsequent complications of post-thrombotic syndrome (PTS), are significant causes of morbidity and mortality in patients. Clinical risk factors for VTE include cancer, major trauma, surgery, sepsis, inflammatory bowel disease, paralysis, prolonged periods of immobility, and aging. Abnormalities in venous blood flow or stasis initiates the activation of endothelial cells, and in concert with platelets, neutrophils and monocytes, propagates VTE in an intact vein. In addition, inflammatory cells play crucial roles in thrombus recanalization and restoration of blood flow via fibrinolysis and vascular remodeling. Faster resolution of the thrombus is key for improved disease prognosis. While in the clinical setting, anticoagulation therapy is successful in preventing propagation of venous thrombi, current therapies are not designed to inhibit inflammation, which can lead to the development of PTS. Animal models of DVT have provided many insights into the molecular and cellular mechanisms involved in the formation, propagation, and resolution of venous thrombi as well as the roles of key components of the fibrinolytic system in these processes. Here, we review the recent advances in our understanding of fibrinolysis and inflammation in the resolution of VTE.

Aggregation of amylin: Spectroscopic investigation.

Apart from its relevance to pathology, protein misfolding disease like Type-II Diabetes Mellitus, caused by amyloids of amylin protein has attracted more attention due to structural changes occurring during the aggregation process. We report extensive spectroscopy data of amylin during fibril formation through Raman, FTIR, CD, UV-vis absorption and photoluminescence (PL) spectroscopy. UV-vis and PL spectrum showed the sigmoidal growth of fibril with a lag time of ~2 days, which is consistent with earlier reported work using dynamic light scattering (DLS). Raman spectra revealed the formation of parallel and anti-parallel ß-sheet from 0% to 20% with ageing (1st day to 21st day) at pH 6.5 ±â€¯0.1. The results are corroborated by CD and FTIR data. These show the change in ß-sheet by 23% at pH 6.5 ±â€¯0.1, 26% at pH = 1.0 ±â€¯0.1 and 30% at pH = 12 ±â€¯0.1. It is also shown that the formation and conversion of other secondary structures into ß-sheet is very sensitive towards the pH and ageing. The study may be used for the development of therapeutic strategies that could inhibit or even reverse the process of aggregation.

PRSS21/testisin inhibits ovarian tumor metastasis and antagonizes proangiogenic angiopoietins ANG2 and ANGPTL4.

Ovarian cancer is the leading cause of death among all the gynecological cancers in the USA. Ovarian cancer employs a unique mode of metastasis, as exfoliated tumor cells disseminate within the peritoneal cavity, colonizing in several sites as well as accumulating ascites. Tumor recurrence and widespread metastasis are significant factors contributing to poor prognosis. PRSS21 is a metastasis-associated ovarian cancer gene that encodes the glycosyl-phosphatidylinositol-linked serine protease, testisin. Testisin expression is increased in multiple ovarian tumor types, with relatively little expression in normal tissues, but is differentially decreased in metastatic ovarian serous carcinomas compared to primary tumors. Here we explored the function of testisin in late-stage ovarian cancer progression using a murine xenograft model of ovarian intraperitoneal tumor metastasis. Increased tumor testisin expression inhibited intra-peritoneal tumor seeding and colonization, ascites accumulation, and metastatic tumor burden that was dependent on catalytically active testisin. The known testisin substrate, protease-activated receptor-2 (PAR-2), is a target of testisin activity. Gene profiling and mechanistic studies demonstrate that testisin activity suppresses the synthesis and secretion of pro-angiogenic angiopoietins, ANG2 and ANGPTL4, which normally promote vascular leak and edema. These observations support a model wherein testisin activates PAR-2 to antagonize proangiogenic angiopoietins that modulate vascular permeability and ascites accumulation associated with ovarian tumor metastasis. KEY MESSAGES: Testisin inhibits metastatic ovarian tumor burden and ascites production. Testisin activity antagonizes ANG2 and ANGPTL4 synthesis and secretion. PAR-2 is a proteolytic target of testisin on the surface of ovarian cancer cells.

Membrane-Anchored Serine Proteases and Protease-Activated Receptor-2-Mediated Signaling: Co-Conspirators in Cancer Progression.

Pericellular proteolysis provides a significant advantage to developing tumors through the ability to remodel the extracellular matrix, promote cell invasion and migration, and facilitate angiogenesis. Recent advances demonstrate that pericellular proteases can also communicate directly to cells by activation of a unique group of transmembrane G-protein-coupled receptors (GPCR) known as protease-activated receptors (PAR). In this review, we discuss the specific roles of one of four mammalian PARs, namely PAR-2, which is overexpressed in advanced stage tumors and is activated by trypsin-like serine proteases that are highly expressed or otherwise dysregulated in many cancers. We highlight recent insights into the ability of different protease agonists to bias PAR-2 signaling and the newly emerging evidence for an interplay between PAR-2 and membrane-anchored serine proteases, which may co-conspire to promote tumor progression and metastasis. Interfering with these pathways might provide unique opportunities for the development of new mechanism-based strategies for the treatment of advanced and metastatic cancers.

Repulsive interaction induces fibril formation and their growth.

In a type-II diabetes disease, amylin protein takes an incorrect structure that leads to the formation of the amyloid fibril. The conversion mechanism of amyloid fibril is not well understood. We have observed a repulsive interaction, in terms of second virial co-efficient (A2), between protein molecules in their native state in the PBS buffer through laser light scattering technique. The A2 switches from repulsive (positive A2) to attractive (negative A2) interactions with elapsed time favoring the formation and growth of the fibril. We report aggregation and fibril growth kinetics of amylin protein in different environmental conditions. The measurement of shape factor (ρ) through light scattering experiment shows a transition from coil-like structure to rod-like growth. In addition to rod-like growth, sheet-like growth of fibril is also observed through analytical and high-resolution TEM imaging techniques. The nucleation leading to elongation of fibrils as well as stacking of individual fibril perpendicular to the fibril axis is held by hydrogen bonding observed through high-resolution TEM.

Improving Cancer Detection and Treatment with Liquid Biopsies and ptDNA.

Liquid biopsy, or the capacity to noninvasively isolate and analyze plasma tumor DNA (ptDNA) using blood samples, represents an important tool for modern oncology that enables increasingly safe, personalized, and robust cancer diagnosis and treatment. Here, we review advances in the development and implementation of liquid biopsy approaches, and we focus on the capacity of liquid biopsy to noninvasively detect oncological disease and enhance early detection strategies. In addition to noting the distinctions between mutation-targeted and mutation-agnostic approaches, we discuss the potential for genomic analysis and longitudinal testing to identify somatic lesions early and to guide intervention at more manageable disease stages.

Hydrophobic hydration and anomalous diffusion of elastin in an ethanolic solution.

Elastin is an important structural protein that confers elasticity to tissues. It is widely used in the biosynthesis of human elastic tissues and exhibits interesting properties. This study reports an insight into the unusual dispersion and anomalous diffusion of elastin in an ethanolic solution. Due to its complex hydrophobic structure, its dispersibility was found to be sensitive towards the hydrophobicity of the solvent. Electrophoresis measurements (zeta-potential data) revealed that its net polarity changed from an anionic to a cationic state with the decreasing solvent hydrophobicity (ethanol content in the solvent). An interesting transition temperature of ∼297 K was observed above which the hydrophobic interactions among the protein molecules became dominant. Double-layer repulsion between protein molecules competes with attractive hydrophobic interactions and causes molecular self-organization. A DLVO-based theoretical model showed that hydrophobic interactions were facilitated by a binary solvent (ethanol-water), and the repulsive double layer screening provided sufficient energy to overcome the interactions between hydrophobic domains in the protein molecule and allow the self-assembly to occur.

Histone deacetylase inhibitors decrease NHEJ both by acetylation of repair factors and trapping of PARP1 at DNA double-strand breaks in chromatin.

Histone deacetylase inhibitors (HDACi) induce acetylation of histone and non-histone proteins, and modulate the acetylation of proteins involved in DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is one of the main pathways for repairing DSBs. Decreased NHEJ activity has been reported with HDACi treatment. However, mechanisms through which these effects are regulated in the context of chromatin are unclear. We show that pan-HDACi, trichostatin A (TSA), causes differential acetylation of DNA repair factors Ku70/Ku80 and poly ADP-ribose polymerase-1 (PARP1), and impairs NHEJ. Repair effects are reversed by treatments with p300/CBP inhibitor C646, with significantly decreased acetylation of PARP1. In keeping with these findings, TSA treatment significantly increases PARP1 binding to DSBs in chromatin. Notably, AML patients treated with HDACi entinostat (MS275) in vivo also show increased formation of poly ADP-ribose (PAR) that co-localizes with DSBs. Further, we demonstrate that PARP1 bound to chromatin increases with duration of TSA exposure, resembling PARP "trapping". Knockdown of PARP1 inhibits trapping and mitigates HDACi effects on NHEJ. Finally, combination of HDACi with potent PARP inhibitor talazoparib (BMN673) shows a dose-dependent increase in PARP "trapping", which correlates with increased apoptosis. These results provide a mechanism through which HDACi inhibits deacetylation and increases binding of PARP1 to DSBs, leading to decreased NHEJ and cytotoxicity of leukemia cells.

An organelle-exclusion envelope assists mitosis and underlies distinct molecular crowding in the spindle region.

The mitotic spindle is a microtubular assembly required for chromosome segregation during mitosis. Additionally, a spindle matrix has long been proposed to assist this process, but its nature has remained elusive. By combining live-cell imaging with laser microsurgery, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy in Drosophila melanogaster S2 cells, we uncovered a microtubule-independent mechanism that underlies the accumulation of molecules in the spindle region. This mechanism relies on a membranous system surrounding the mitotic spindle that defines an organelle-exclusion zone that is conserved in human cells. Supported by mathematical modeling, we demonstrate that organelle exclusion by a membrane system causes spatio-temporal differences in molecular crowding states that are sufficient to drive accumulation of mitotic regulators, such as Mad2 and Megator/Tpr, as well as soluble tubulin, in the spindle region. This membranous "spindle envelope" confined spindle assembly, and its mechanical disruption compromised faithful chromosome segregation. Thus, cytoplasmic compartmentalization persists during early mitosis to promote spindle assembly and function.