Ninein is a multifunctional protein involved in microtubule (MT) organization and dynein/dynactin complex recruitment and activation. Several isoforms of ninein have been identified in various tissues, however, their relative contribution(s) are not clear. Here, we identify two ninein isoforms in mouse macrophages with distinct C-termini and disproportionate expression levels; a canonical ninein (nineinCAN) isoform and ninein isoform 2 (nineinISO2). Analysis of ninein pre-mRNA exon-intron boundaries revealed that nineinISO2 transcript is likely generated by two alternative splicing site selection events predicted to result in a distinct 3D structure compared to nineinCAN. We used selective and total protein knockdown experiments to assess the intracellular and functional roles of ninein in macrophages. Live cell imaging analyses of macrophages implicated both isoforms in regulating cell proliferation. MT regrowth following nocodazole depolymerization showed that both isoforms contributed to MT nucleation and structural integrity of the centrosome, as cells lacking nineinCAN or nineinISO2 contained multiple ectopic γ-tubulin foci. However, nineinCAN, but not nineinISO2, was important for the separation of duplicated centrosomes during cell division. Despite a requirement of both ninein isoforms to recruit dynein/dynactin to the centrosome, only nineinCAN was required for Golgi positioning and morphology, dynein-dependent events. We additionally found that nineinISO2 was the primary isoform required for F-actin recruitment during the internalization of IgG-opsonized particles. Our study indicates that alternative splicing promotes both redundant and differential activities for ninein in MT organization, organelle positioning, and macrophage function.

The bone is a highly dynamic organ that undergoes continuous remodeling through an intricate balance of bone formation and degradation. Hyperactivation of the bone-degrading cells, the osteoclasts (OCs), occurs in disease conditions and hormonal changes in females, resulting in osteoporosis, a disease characterized by altered microarchitecture of the bone tissue, and increased bone fragility. Thus, building robust assays to quantify OC resorptive activity to examine the molecular mechanisms underlying bone degradation is critical. Here, we establish an in vitro model to investigate the effect of estrogen withdrawal on OCs derived from the mouse macrophage RAW 264.7 cell line in a bone biomimetic microenvironment. This simple and robust model can also be adapted to examine the effect of drugs and genetic factors influencing OC resorptive activity in addition to being compatible with fluorescent imaging.

Tubulinopathies and neurodevelopmental ciliopathies are two groups of genetic disorders that cause structural brain malformations. Tubulinopathies result from mutations in tubulins, the building blocks of microtubules, most of which are dominant. Neurodevelopmental ciliopathies are mostly recessive disorders caused by defects in the primary cilium, an organelle that regulates key signaling pathways during brain development. Although more than 40 genes have been associated with neurodevelopmental ciliopathies, many patients still lack a known genetic cause. Here, we present a de novo heterozygous missense variant (p.G308S) in Tubulin Beta Class I (TUBB) identified in a patient showing features of both ciliopathy and tubulinopathy. While microtubules are essential for primary cilia formation and function, an association between tubulin mutations and neurodevelopmental ciliopathies has not been established. Using patient-derived cells and gene-edited isogenic cell lines, we show that the identified variant impairs the early stages of cilia formation by altering microtubule dynamics and structure. Consistent with this, mice carrying the variant display decreased ciliation in the cerebellum and kidney. Furthermore, we demonstrate that the disease mechanism is not haploinsufficiency and that other patient mutations in TUBB affect cilia formation, putting forward defective ciliogenesis as a contributing pathogenic factor in a subset of tubulinopathy patients.

Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

Phagocytosis by macrophages is a highly polarized process to destroy large target cells. Binding to particles induces extensive cortical actin-generated forces that drive the formation of elaborate pseudopods around the target particle. Postinternalization, the resultant phagosome is driven toward the cell interior on microtubules (MTs) by cytoplasmic dynein. However, it is unclear whether dynein and cargo-adaptors contribute to the earlier steps of particle internalization and phagosome formation. Here we reveal that ninein, a MT minus-end-associated protein that localizes to the centrosome, is also present at the phagocytic cup in macrophages. Ninein depletion impairs particle internalization by delaying the early F-actin recruitment to sites of particle engagement and cup formation, with no impact on F-actin dynamics beyond this initial step. Ninein forms membrane-bound clusters on phagocytic cups that do not nucleate acentrosomal MTs but instead mediate the assembly of dynein-dynactin complex at active phagocytic membranes. Both ninein depletion and pharmacological inhibition of dynein activity reduced inward displacement of bound particles into macrophages. We found that ninein and dynein motor activity were required for timely retrograde movement of phagosomes and for phagolysosome formation. Taken together, these data show that ninein, alone and with dynein, play significant roles during phagocytosis.

Introduction: Legionella is a gram-negative bacterium that replicates intracellularly within macrophages. Legionella utilizes effector proteins to hijack ER-Golgi vesicle trafficking to sustain proliferation in its intracellular niche. Legionella has a considerable influence on O-glycosylation but not N-glycosylation events in the Golgi of infected cells. This research aims to optimize the use of fluorescent lectins, which are proteins that bind carbohydrates, to effectively label host-cell glycocalyx during Legionella infection. Methods: Epifluorescence imaging or flow cytometry were used to optimize the lectin staining methodology. We noted that the most effective conditions for lectin-labeling were when live HeLa cells were incubated with lectins diluted in Hank’s balanced salt solution (HBSS) with 3% Bovine serum albumin (BSA) for 10-30 minutes at 4 °C. Results: Incubating suspended cells with lectins necessitated smaller lectin concentrations, whereas lectin labeling of adherent cells required considerably larger concentrations. Wheat germ agglutinin (WGA) lectin mean fluorescence intensity (MFI) was concentration-dependent, but Concanavalin A (ConA) and Maclura pomifera (MPA) MFIs did not alter substantially with increasing lectin concentrations. Discussion: The optimal lectin concentration required was lectin-specific and based on whether the lectin fluorescence was assessed using flow cytometry or epifluorescence. Furthermore, the use of phosphate-buffered saline (PBS) for lectin dilution, cell permeabilization for intracellular labelling, and incubation of lectins in fixed cells reduced productive labelling of lectins on cell surfaces because it inhibited the lectin's ability to effectively bind the associated carbohydrate structure. Conclusion: Further research using diverse lectins on U937 macrophages is necessary to reach a definitive conclusion on the effect of Legionella on the overall host-cell glycocalyx composition during infection of these relevant immune cells.

ABSTRACT Tubulinopathies and neurodevelopmental ciliopathies are two groups of genetic disorders characterized by abnormal brain development resulting in structural brain malformations. Tubulinopathies are caused by dominant missense mutations in genes encoding for tubulins, the building blocks of microtubules. Neurodevelopmental ciliopathies are mostly recessive disorders caused by defects in the function of the primary cilium, a sensory organelle that modulates signaling pathways important for brain development. Though more than 40 genes have been associated with neurodevelopmental ciliopathies, many patients still do not have an identified genetic etiology. Herein, we present a novel de novo heterozygous missense variant in Tubulin Beta Class I ( TUBB ) identified through whole-genome sequencing analysis in a patient with both ciliopathy and tubulinopathy brain features. While microtubules are fundamental to primary cilia formation and function, no association between mutations in tubulin genes and neurodevelopmental ciliopathies has been found to date. Using patient-derived cells and gene-edited isogenic cell lines, we show that the identified variant impairs the early stages of cilia formation by altering microtubule dynamics and structure. Furthermore, we demonstrate that the disease mechanism is not haploinsufficiency and that other patient mutations in TUBB affect cilia formation in vitro , putting forward defective ciliogenesis as a contributing pathogenic factor in a subset of tubulinopathy patients.

Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER–Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.

Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of the dense, calcified bone matrix. Microtubules (MTs) contribute to the polarization and trafficking events involved in bone resorption by osteoclasts; however, the origin of these elaborate arrays is less clear. Osteoclasts arise through cell fusion of precursor cells. Previous studies have suggested that centrosome MT nucleation is lost during this process, with the nuclear membrane and its surrounding Golgi serving as the major MT organizing centers (MTOCs) in these cells. Here we reveal that precursor cell centrosomes are maintained and functional in the multinucleated osteoclast and interestingly form large MTOC clusters, with the clusters organizing significantly more MTs compared with individual centrosomes. MTOC cluster formation requires dynamic MTs and minus-end directed MT motor activity. Inhibition of these centrosome clustering elements had a marked impact on both F-actin ring formation and bone resorption. Together these findings show that multinucleated osteoclasts employ unique centrosomal clusters to organize the extensive MTs during bone attachment and resorption.

Phagocytosis is an essential process for the uptake of large (>0.5 µm) particulate matter including microbes and dying cells. Specialized cells in the body perform phagocytosis which is enabled by cell surface receptors that recognize and bind target cells. Professional phagocytes play a prominent role in innate immunity and include macrophages, neutrophils and dendritic cells. These cells display a repertoire of phagocytic receptors that engage the target cells directly, or indirectly via opsonins, to mediate binding and internalization of the target into a phagosome. Phagosome maturation then proceeds to cause destruction and recycling of the phagosome contents. Key subsequent events include antigen presentation and cytokine production to alert and recruit cells involved in the adaptive immune response. Bridging the innate and adaptive immunity, macrophages secrete a broad selection of inflammatory mediators to orchestrate the type and magnitude of an inflammatory response. This review will focus on cytokines produced by NF-κB signaling which is activated by extracellular ligands and serves a master regulator of the inflammatory response to microbes. Macrophages secrete pro-inflammatory cytokines including TNFα, IL1β, IL6, IL8 and IL12 which together increases vascular permeability and promotes recruitment of other immune cells. The major anti-inflammatory cytokines produced by macrophages include IL10 and TGFβ which act to suppress inflammatory gene expression in macrophages and other immune cells. Typically, macrophage cytokines are synthesized, trafficked intracellularly and released in response to activation of pattern recognition receptors (PRRs) or inflammasomes. Direct evidence linking the event of phagocytosis to cytokine production in macrophages is lacking. This review will focus on cytokine output after engagement of macrophage phagocytic receptors by particulate microbial targets. Microbial receptors include the PRRs: Toll-like receptors (TLRs), scavenger receptors (SRs), C-type lectin and the opsonic receptors. Our current understanding of how macrophage receptor stimulation impacts cytokine production is largely based on work utilizing soluble ligands that are destined for endocytosis. We will instead focus this review on research examining receptor ligation during uptake of particulate microbes and how this complex internalization process may influence inflammatory cytokine production in macrophages.

The mechanism and role of transient F-actin recruitment, or F-actin 'flashes', on phagosomes remains enigmatic. Here we provide a comprehensive characterization of F-actin flashing dynamics on phagosomes, including receptor and signaling involvement. F-actin flashes predominate during the integrin-driven complement receptor (CR)-mediated phagocytosis. F-actin flashes begin shortly after internalization and persist on phagosomes for approximately 3 minutes before disassembling and reassembling several times within the first hour. Strikingly, the appearance of F-actin flashes on phagosomes coincides with morphological deformation, lysis and occasional fission of internalized red blood cells. The cadence of flashes depends on particle stiffness, and the F-actin networks on phagosomes are enriched in mechanosensitive components including focal adhesion proteins, RhoA and actomyosin. Inhibiting Arp2/3 and myosin IIA activity significantly reduces the frequency at which phagosome cargo becomes deformed during transient F-actin accumulation. At later time points, post-F-actin flashing, enhanced degradation of phagosome contents is observed, compared with non-flashing phagosomes. Taken together, these data suggest that actomyosin-driven phagosome contractions serve to disrupt malleable particles physically, a process akin to mastication, to enhance later enzymatic digestion.

Astronauts are prone to a condition known as disuse osteoporosis as the microgravity environment negates the need for skeletal weight bearing. Recently, the development of new strategies to study bone loss in microgravity has been advancing at a rapid pace. As a result, several emerging technologies have paved the way for new research into the cellular and physiological mechanisms involved in disuse osteoporosis. In this review, we discuss the most impactful and current methodologies and technologies for both in vivo and in vitro studies of bone loss in space and with simulators on Earth from the past decade. We cover research performed on the International Space Station, uncrewed satellites, head-down tilt bed rest, rodent hindlimb unloading, and 2D/3D clinorotation for cell culture which are all established methods to mechanically unload the skeleton and/or bone cells. We also summarize the experimental findings documenting the changes that occur following exposure to unloading on a macroscopic scale, such as morphometric changes to the bone structure, and on the microscopic scale, such as effects on bone-forming osteoblasts, bone-resorbing osteoclasts, and mechanical stress-sensing osteocytes.

Purpose: The rotary cell culture system (RCCS) is a common clinorotation device for cell culture. It is also used as a low-shear suspension culture bioreactor to form functionalized 3D tissue constructs and to model microgravity. We sought to develop a 3D scaffold composed of type I collagen and hydroxyapatite (collagen-HA) to characterize MLO-Y4 osteocytes following suspension culture or clinorotation. Materials and Methods: MLO-Y4 cells were embedded in collagen-HA. The scaffold was formed into droplets for suspension culture or wall-adhered to the RCCS for clinorotation. AFM, rheometry, immunofluorescence and qRT-PCR were employed to measure the scaffold stiffness, cell viability and gene expression of cells in collagen-HA scaffolds. Dendritic cells were visualized and quantified and gene expression after suspension culture and clinorotation was compared to static controls. Results: The optimized scaffold for the RCCS consisted of collagen with 6 mg/mL HA which had a stiffness of < 1 kPa. MLO-Y4 cell viability was higher in collagen-HA scaffolds, compared to scaffolds without HA. Collagen-HA scaffolds induced higher osteocyte-specific gene expression compared to cells cultured on 2D plastic. Cells in the scaffold downregulated DMP1, E11, IL-6, and RANKL, and had fewer dendritic cells following suspension culture whereas clinorotation downregulated DMP1 and E11 genes, compared to static controls. Conclusions: Suspension culture for 3 days in collagen-HA stimulates growth of osteocytes but may also desensitize them to mechanical cues. Clinorotation for 3 days in collagen-HA does not stimulate proliferation or expression of mechanosensitive genes, indicating that it may be an effective mechanical unloading environment.

Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1β, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.

Patients with rheumatoid arthritis (RA) have very different outcomes, particularly with regard to bone erosions. Since osteoclasts are responsible for bone destruction adjacent to rheumatoid synovium, profiling osteoclasts from circulating precursors in RA could help identify patients at risk for bone destruction. In this study, we sought to determine whether the functional characteristics of osteoclasts generated from their blood precursors were modified by RA activity or were intrinsic to osteoclasts and associated with the RA phenotype (erosive or not).Osteoclasts were generated in vitro from peripheral blood mononuclear cells (PBMCs) of subjects with RA (n = 140), as well as sex- and age-matched healthy controls (n = 101). Osteoclastic parameters were analyzed at baseline and during the follow-up for up to 4 years, with regular assessment of RA activity, bone erosions, and bone mineral density (BMD). As a validation cohort, we examined RA patients from the Early Undifferentiated PolyArthritis (EUPA) study (n = 163).The proportion of CD14+ PBMC was higher in RA than in control subjects, but inversely correlated with the 28-joint disease activity score (DAS28). Also surprisingly, in osteoclast cultures from PBMCs, active RA was associated with lower osteoclastogenic capacity, while in vitro bone resorption per osteoclast and resistance to apoptosis were similar in both active and quiescent RA. In a small subgroup analysis, osteoclasts from subjects with recent RA that had progressed at four years to an erosive RA exhibited at baseline greater resistance to apoptosis than those from patients remaining non-erosive.Our findings establish that when RA is active, circulating monocytes have a reduced potential to generate osteoclasts from PBMCs in vitro. In addition, osteoclasts associated with erosive disease had resistance to apoptosis from the start of RA.

Our current understanding of phagocytosis is largely derived from studies of individual receptor-ligand interactions and their downstream signaling pathways. Because phagocytes are exposed to a variety of ligands on heterogeneous target particles in vivo, it is important to observe the engagement of multiple receptors simultaneously and the triggered involvement of downstream signaling pathways. Potential crosstalk between the two well-characterized opsonic receptors, FcγR and CR3, was briefly explored in the early 1970s, where macrophages were challenged with dual-opsonized targets. However, subsequent studies on receptor crosstalk were primarily restricted to using single opsonins on different targets, typically at saturating opsonin conditions. Beyond validating these initial explorations on receptor crosstalk, we identify the early signaling mechanisms that underlie the binding and phagocytosis during the simultaneous activation of both opsonic receptors, through the presence of a dual-opsonized target (immunoglobulin G [IgG] and C3bi), compared with single receptor activation. For this purpose, we used signaling protein inhibitor studies as well as live cell brightfield and fluorescent imaging to fully understand the role of tyrosine kinases, F-actin dynamics and internalization kinetics for FcγR and CR3. Importantly, opsonic receptors were studied together and in isolation, in the context of sparsely opsonized targets. We observed enhanced particle binding and a synergistic effect on particle internalization during the simultaneous activation of FcγR and CR3 engaged with sparsely opsonized targets. Inhibition of early signaling and cytoskeletal molecules revealed a differential involvement of Src kinase for FcγR- vs CR3- and dual receptor-mediated phagocytosis. Src activity recruits Syk kinase and we observed intermediate levels of Syk phosphorylation in dual-opsonized particles compared with those opsonized with IgG or C3bi alone. These results likely explain the intermediate levels of F-actin that is recruited to sites of dual-opsonized particle uptake and the notoriously delayed internalization of C3bi-opsonized targets by macrophages.

Macropinocytosis or “cell drinking” involves the elaboration of membrane ruffles that enclose and internalize extracellular fluids. Using lattice light sheet microscopy, Condon et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201804137) reveal the presence of parallel membrane protrusions termed “tent poles” that flank and direct membrane ruffle formation.

Bone loss is a major health problem that many aging individuals will face and thus research focusing on enhancing bone formation is of great importance. Cell biology or in vitro studies are particularly useful in exploring the exact effects a vitamin, supplement or drug has on particular processes within a certain cell type. Although there have been many cell biology articles focusing on the effects of vitamin D, K1 or K2 addition on bone formation in vitro, there has yet to be a consensus amongst the literature. The purpose of this article is to determine the effects of vitamin D, K1 and K2 supplementation on osteoblast maturation parameters through meta-analysis of past cell biology literature. A Hedges d effect size was calculated for each experiment extracted from past literature and the experiments were grouped by experiment and cell type. Homogeneity was assessed by the Cochran’s Q test, while the effect sizes’ departure from zero was assessed by a 95% confidence interval and a non-directional test. Supplementation with vitamin D, K1 and K2, along with the combination of vitamin K2+ 1,25-dihydroxyvitamin D, increased bone mineralization, while not consistently affecting all of the other parameters associated with bone formation. Vitamin K2 and D addition had variable effects on bone formation using different cell types, which calls into question the suitability of particular cell lines as models for clinical trials. Therefore, the conditions and parameters in which bone formation is studied in vitro must be considered carefully before running a vitamin supplementation or drug-testing experiment.

During the cell cycle, genetic materials and organelles are duplicated to ensure that there is sufficient cellular content for daughter cells. While Golgi growth in interphase has been observed in lower eukaryotes, the elaborate ribbon structure of the mammalian Golgi apparatus has made it challenging to monitor. Here we demonstrate the growth of the mammalian Golgi apparatus in its protein content and volume during interphase. Through ultrastructural analyses, physical growth of the Golgi apparatus was revealed to occur by cisternal elongation of the individual Golgi stacks. By examining the timing and regulation of Golgi growth, we established that Golgi growth starts after passage through the cell growth checkpoint at late G(1) phase and continues in a manner highly correlated with cell size growth. Finally, by identifying S6 kinase 1 as a major player in Golgi growth, we revealed the coordination between cell size and Golgi growth via activation of the protein synthesis machinery in early interphase.