Abemaciclib induces atypical cell death in cancer cells characterized by formation of cytoplasmic vacuoles derived from lysosomes.

In the cell cycle, the G1 /S transition is controlled by the cyclin-dependent kinase (CDK) 4/6-cyclin D complex. Constitutive activation of CDK4/6 dysregulates G1 /S transition, leading to oncogenic transformation. We found that 3 CDK4/6 inhibitors, abemaciclib, ribociclib, and palbociclib, exerted a cytocidal effect as well as a cytostatic effect at the G1 phase in cancer cell lines, including A549 human non-small cell lung cancer cells. Among these inhibitors, abemaciclib exhibited the most potent cytotoxic effect. The cell-death phenotype induced by abemaciclib, which entailed formation of multiple cytoplasmic vacuoles, was not consistent with apoptosis or necroptosis. Abemaciclib blocked autophagic flux, resulting in accumulation of autophagosomes, however vacuole formation and cell death induced by abemaciclib were independent of autophagy. In addition, methuosis, a cell-death phenotype characterized by vacuole formation induced by excessive macropinocytosis, was excluded because the vacuoles did not incorporate fluorescent dextran. Of note, both formation of vacuoles and induction of cell death in response to abemaciclib were inhibited by vacuolar-type ATPase (V-ATPase) inhibitors such as bafilomycin A1 and concanamycin A. Live-cell imaging revealed that the abemaciclib-induced vacuoles were derived from lysosomes that expanded following acidification. Transmission electron microscopy revealed that these vacuoles contained undigested debris and remnants of organelles. Cycloheximide chase assay revealed that lysosomal turnover was blocked by abemaciclib. Furthermore, mTORC1 inhibition along with partial lysosomal membrane permeabilization occurred after abemaciclib treatment. Together, these results indicate that, in cancer cells, abemaciclib induces a unique form of cell death accompanied by swollen and dysfunctional lysosomes.

Factors That Affect the Enlargement of Bacterial Protoplasts and Spheroplasts.

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.

Dynamic organelle changes and autophagic processes in lily pollen germination.

Pollen germination is a crucial process in the life cycle of flowering plants, signifying the transition of quiescent pollen grains into active growth. This study delves into the dynamic changes within organelles and the pivotal role of autophagy during lily pollen germination. Initially, mature pollen grains harbor undifferentiated organelles, including amyloplasts, mitochondria, and the Golgi apparatus. However, germination unveils remarkable transformations, such as the redifferentiation of amyloplasts accompanied by starch granule accumulation. We investigate the self-sustained nature of amylogenesis during germination, shedding light on its association with osmotic pressure. Employing BODIPY 493/503 staining, we tracked lipid body distribution throughout pollen germination, both with or without autophagy inhibitors (3-MA, NEM). Typically, lipid bodies undergo polarized movement from pollen grains into elongating pollen tubes, a process crucial for directional growth. Inhibiting autophagy disrupted this essential lipid body redistribution, underscoring the interaction between autophagy and lipid body dynamics. Notably, the presence of tubular endoplasmic reticulum (ER)-like structures associated with developing amyloplasts and lipid bodies implies their participation in autophagy. Starch granules, lipid bodies, and membrane remnants observed within vacuoles further reinforce the involvement of autophagic processes. Among the autophagy inhibitors, particularly BFA, significantly impede germination and growth, thereby affecting Golgi morphology. Immunogold labeling substantiates the pivotal role of the ER in forming autophagosome-like compartments and protein localization. Our proposed speculative model of pollen germination encompasses proplastid differentiation and autophagosome formation. This study advances our understanding of organelle dynamics and autophagy during pollen germination, providing valuable insights into the realm of plant reproductive physiology.

Cellular and physiological functions of SGR family in gravitropic response in higher plants.

BACKGROUND: In plants, gravity directs bidirectional growth; it specifies upward growth of shoots and downward growth of roots. Due to gravity, roots establish robust anchorage and shoot, which enables to photosynthesize. It sets optimum posture and develops plant architecture to efficiently use resources like water, nutrients, CO2, and gaseous exchange. Hence, gravitropism is crucial for crop productivity as well as for the growth of plants in challenging climate. Some SGR members are known to affect tiller and shoot angle, organ size, and inflorescence stem in plants. AIM OF REVIEW: Although the SHOOT GRAVITROPISM (SGR) family plays a key role in regulating the fate of shoot gravitropism, little is known about its function compared to other proteins involved in gravity response in plant cells and tissues. Moreover, less information on the SGR family's physiological activities and biochemical responses in shoot gravitropism is available. This review scrutinizes and highlights the recent developments in shoot gravitropism and provides an outlook for future crop development, multi-application scenarios, and translational research to improve agricultural productivity. KEY SCIENTIFIC CONCEPTS OF REVIEW: Plants have evolved multiple gene families specialized in gravitropic responses, of which the SGR family is highly significant. The SGR family regulates the plant's gravity response by regulating specific physiological and biochemical processes such as transcription, cell division, amyloplast sedimentation, endodermis development, and vacuole formation. Here, we analyze the latest discoveries in shoot gravitropism with particular attention to SGR proteins in plant cell biology, cellular physiology, and homeostasis. Plant cells detect gravity signals by sedimentation of amyloplast (starch granules) in the direction of gravity, and the signaling cascade begins. Gravity sensing, signaling, and auxin redistribution (organ curvature) are the three components of plant gravitropism. Eventually, we focus on the role of multiple SGR genes in shoot and present a complete update on the participation of SGR family members in gravity.

Novobiocin inhibits membrane synthesis and vacuole formation of Enterococcus faecalis protoplasts.

We demonstrate that plasma membrane biosynthesis and vacuole formation require DNA replication in Enterococcus faecalis protoplasts. The replication inhibitor novobiocin inhibited not only DNA replication but also cell enlargement (plasma membrane biosynthesis) and vacuole formation during the enlargement of the E. faecalis protoplasts. After novobiocin treatment prior to vacuole formation, the cell size of E. faecalis protoplasts was limited to 6 µm in diameter and the cells lacked vacuoles. When novobiocin was added after vacuole formation, E. faecalis protoplasts grew with vacuole enlargement; after novobiocin removal, protoplasts were enlarged again. Although cell size distribution of the protoplasts was similar following the 24 h and 48 h novobiocin treatments, after 72 h of novobiocin treatment there was a greater number of smaller sized protoplasts, suggesting that extended novobiocin treatment may inhibit the re-enlargement of E. faecalis protoplasts after novobiocin removal. Our findings demonstrate that novobiocin can control the enlargement of E. faecalis protoplasts due to inhibition of DNA replication.

Enhanced expression and distinctive characterization of a long-acting FGF21 and its potential to alleviate nonalcoholic steatohepatitis.

We have previously constructed a novel polypeptide, PsTag, that should be useful in the development of biologics with properties comparable to those achievable by PEGylation, but with potentially less side effects. However, the low fermentation yields of polypeptide fusion proteins may limit the application of this technology. We suspected that when polypeptide fusion protein was expressed in E. coli, the corresponding 8 tRNAs were needed to transport a large number of repetitive 5 amino acids to the ribosomes and thus, resulting in a relative deficiency of these tRNAs. PsTag600-FGF21, a long-acting FGF21 fusion protein, was used as a model for studying the effects of these non-rare tRNAs on the efficiency of heterologous protein production in E. coli. To further enhance the expression level and facilitate purification, secretory expressions of PsTag600-FGF21 were achieved by fusion with three signal peptides. Meanwhile, a comparison of several distinctive characterizations was carried out between PsTag600-FGF21 and PEG20K-FGF21. We investigated the protective effects of PsTag600-FGF21 in a nonalcoholic steatohepatitis model induced by methionine- and choline-deficient diet. Our results showed that the provision of 8 tRNAs and secretory expression remarkably increased the expression levels of PsTag fusion protein, meanwhile there were no significant effects on E. coli growth states. PsTag600-FGF21 had a larger hydrodynamic volume, a higher affinity and a longer plasma half-life than PEG20K-FGF21, while avoiding vacuole formation in mice. In NASH mice, administration of PsTag600-FGF21 reduced hepatic steatosis, fibrosis and inflammation. Therefore, PsTag600-FGF21 with higher expression level may be further developed for potentially application in clinics.

Alterations in the sugar metabolism and in the vacuolar system of mesophyll cells contribute to the desiccation tolerance of Haberlea rhodopensis ecotypes.

Haberlea rhodopensis belongs to the small group of resurrection plants having the unique ability to survive desiccation to air dry state retaining most of its chlorophyll content and then resume normal function upon rehydration. It prefers the shady valleys and northward facing slopes of limestone ridges in mountain zones with high average humidity. Nevertheless, it can be found rarely on rocks directly exposed to the sunlight, without the coverage of the canopy. In the present study, we follow the alterations in the subcellular organization of mesophyll cells and sugar metabolism upon desiccation of shade and sun H. rhodopensis plants. Composition and content of soluble carbohydrates during desiccation and rehydration were different in plants grown below the trees or on the sunny rocks. Sucrose, however, was dominating in both ecotypes. The amount of starch grains in chloroplasts was inversely related to that of sugars. Concomitantly with these changes, the number of vacuoles was multiplied in the cells. This can be explained by the development of small (secondary) vacuoles peripherally in the cytoplasm, rather than by the fragmentation of the single vacuole, proposed earlier in the literature. Accordingly, the centripetal movement of chloroplasts and other organelles may be a result of the dynamic changes in the vacuolar system. Upon rehydration, the inner vacuoles enlarged and the organelles returned to their normal position.

A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication.

Intracellular pathogens manipulate host organelles to support replication within cells. For Legionella pneumophila, the bacterium translocates proteins that establish an endoplasmic reticulum (ER)-associated replication compartment. We show here that the bacterial Sde proteins target host reticulon 4 (Rtn4) to control tubular ER dynamics, resulting in tubule rearrangements as well as alterations in Rtn4 associated with the replication compartment. These rearrangements are triggered via Sde-promoted ubiquitin transfer to Rtn4, occurring almost immediately after bacterial uptake. Ubiquitin transfer requires two sequential enzymatic activities from a single Sde polypeptide: an ADP-ribosyltransferase and a nucleotidase/phosphohydrolase. The ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase, resulting in either transfer of ubiquitin to Rtn4 or phosphoribosylation of ubiquitin in the absence of a ubiquitination target. Therefore, a single bacterial protein drives a multistep biochemical pathway to control ubiquitination and tubular ER function independently of the host ubiquitin machinery.