Mechanisms of intracellular scaling.

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, which places variable metabolic and functional demands on organelles and internal structures. A fundamental question is how essential subcellular components scale to accommodate cell size differences. Nuclear transport has emerged as a conserved means of scaling nuclear size. A meiotic spindle scaling factor has been identified as the microtubule-severing protein katanin, which is differentially regulated by phosphorylation in two different-sized frog species. Anaphase mechanisms and levels of chromatin compaction both act to coordinate cell size with spindle and chromosome dimensions to ensure accurate genome distribution during cell division. Scaling relationships and mechanisms for many membrane-bound compartments remain largely unknown and are complicated by their heterogeneity and dynamic nature. This review summarizes cell and organelle size relationships and the experimental approaches that have elucidated mechanisms of intracellular scaling.

Regulation of organelle size and organization during development.

During early embryogenesis, as cells divide in the developing embryo, the size of intracellular organelles generally decreases to scale with the decrease in overall cell size. Organelle size scaling is thought to be important to establish and maintain proper cellular function, and defective scaling may lead to impaired development and disease. However, how the cell regulates organelle size and organization are largely unanswered questions. In this review, we summarize the process of size scaling at both the cell and organelle levels and discuss recently discovered mechanisms that regulate this process during early embryogenesis. In addition, we describe how some recently developed techniques and Xenopus as an animal model can be used to investigate the underlying mechanisms of size regulation and to uncover the significance of proper organelle size scaling and organization.

Nuclear size is regulated by importin α and Ntf2 in Xenopus.

The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.

Nuclear F-actin and Lamin A antagonistically modulate nuclear shape.

Nuclear shape influences cell migration, gene expression and cell cycle progression, and is altered in disease states like laminopathies and cancer. What factors and forces determine nuclear shape? We find that nuclei assembled in Xenopus egg extracts in the presence of dynamic F-actin exhibit a striking bilobed nuclear morphology with distinct membrane compositions in the two lobes and accumulation of F-actin at the inner nuclear envelope. The addition of Lamin A (encoded by lmna), which is absent from Xenopus eggs, results in rounder nuclei, suggesting that opposing nuclear F-actin and Lamin A forces contribute to the regulation of nuclear shape. Nuclear F-actin also promotes altered nuclear shape in Lamin A-knockdown HeLa cells and, in both systems, abnormal nuclear shape is driven by formins and not Arp2/3 or myosin. Although the underlying mechanisms might differ in Xenopus and HeLa cells, we propose that nuclear F-actin filaments nucleated by formins impart outward forces that lead to altered nuclear morphology unless Lamin A is present. Targeting nuclear actin dynamics might represent a novel approach to rescuing disease-associated defects in nuclear shape.

An insect antifreeze protein from Anatolica polita enhances the cryoprotection of Xenopus laevis eggs and embryos.

Cryoprotection is of interest in many fields of research, necessitating a greater understanding of different cryoprotective agents. Antifreeze proteins have been identified that have the ability to confer cryoprotection in certain organisms. Antifreeze proteins are an evolutionary adaptation that contributes to the freeze resistance of certain fish, insects, bacteria and plants. These proteins adsorb to an ice crystal's surface and restrict its growth within a certain temperature range. We investigated the ability of an antifreeze protein from the desert beetle Anatolica polita, ApAFP752, to confer cryoprotection in the frog Xenopus laevis. Xenopus laevis eggs and embryos microinjected with ApAFP752 exhibited reduced damage and increased survival after a freeze-thaw cycle in a concentration-dependent manner. We also demonstrate that ApAFP752 localizes to the plasma membrane in eggs and embryonic blastomeres and is not toxic for early development. These studies show the potential of an insect antifreeze protein to confer cryoprotection in amphibian eggs and embryos.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/ß-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Has Self-reported Marijuana Use Changed in Patients Undergoing Total Joint Arthroplasty After the Legalization of Marijuana?

BACKGROUND: Marijuana use has become more accessible since its recent legalization in several states. However, its use in a total joint arthroplasty population to our knowledge has not been reported, and the implications of its use in this setting remain unclear. QUESTIONS/PURPOSES: We report (1) the self-reported use of marijuana in patients undergoing total joint arthroplasty both before and after its legalization; and (2) clinical and demographic factors associated with marijuana use in patients undergoing total joint arthroplasty. METHODS: One thousand records of patients undergoing primary total joint arthroplasty (500 consecutive before and 500 consecutive after the legalization of the commercial sale of marijuana in Colorado) were included for analysis. Preoperative medical history and physicals were retrospectively reviewed for self-reported and reasons (medicinal versus recreational) for use. Additionally, patient records were used to determine insurance type, age, gender, smoking status, history of substance abuse, preoperative narcotic use, alcohol intake, and the type of arthroplasty performed. RESULTS: Self-reported use after legalization dramatically increased from 1% (four of 500) to 11% (55 of 500) (odds ratio [OR], 15.3 [95% confidence interval, 5.5-42.6]; p < 0.001) after legalization. For those reporting use after legalization, 46% (25 of 55) of patients reported recreational use, 26% (14 of 55) medicinal use, 27% (15 of 55) did not report a reason for use, and 2% (one of 55) reported both recreational and medicinal use. Factors associated with use included younger age (with a 10-year mean difference between the groups [p < 0.001]), male gender (36 of 59 users [61%] versus 411 of 941 nonusers [44%]; OR, 2.02; p < 0.01), current smokers (22 of 59 users [37%] versus 54 of 941 [6%] nonusers; OR, 0.09; p < 0.01), a history of substance abuse (eight of 59 users [14%] versus 18 of 941 nonusers [2%]; OR, 8.04; p < 0.001), insurance type (Medicaid only, 28 of 59 [48%] users versus 56 of 941 [6%] nonusers; OR, 20.45; p < 0.01), and preoperative narcotic use (eight of 59 users [14%] versus 17 of 941 nonusers [2%]; OR, 2.4; p < 0.001). We did not find differences with regard to alcohol use, amount of alcohol consumption, or insurance types other than Medicaid. CONCLUSIONS: These results suggest the legalization of marijuana has led to either more users or more patients who are willing to report its use given the lack of legal ramifications. Despite these findings, the evidence to date precludes the use of marijuana postoperatively in patients undergoing total joint arthroplasty. Further investigation, ideally in a prospective randomized manner, should focus on opioid consumption, nausea, sleep patterns, and outcomes in patients using marijuana who are undergoing total joint arthroplasty before recommendations can be made for its use. LEVEL OF EVIDENCE: Level III, therapeutic study.

Nuclear size is sensitive to NTF2 protein levels in a manner dependent on Ran binding.

Altered nuclear size is associated with many cancers, and determining whether cancer-associated changes in nuclear size contribute to carcinogenesis necessitates an understanding of mechanisms of nuclear size regulation. Although nuclear import rates generally positively correlate with nuclear size, NTF2 levels negatively affect nuclear size, despite the role of NTF2 (also known as NUTF2) in nuclear recycling of the import factor Ran. We show that binding of Ran to NTF2 is required for NTF2 to inhibit nuclear expansion and import of large cargo molecules in Xenopus laevis egg and embryo extracts, consistent with our observation that NTF2 reduces the diameter of the nuclear pore complex (NPC) in a Ran-binding-dependent manner. Furthermore, we demonstrate that ectopic NTF2 expression in Xenopus embryos and mammalian tissue culture cells alters nuclear size. Finally, we show that increases in nuclear size during melanoma progression correlate with reduced NTF2 expression, and increasing NTF2 levels in melanoma cells is sufficient to reduce nuclear size. These results show a conserved capacity for NTF2 to impact on nuclear size, and we propose that NTF2 might be a new cancer biomarker.

Revision Total Knee Arthroplasty for Arthrofibrosis.

BACKGROUND: Arthrofibrosis after TKA is a significant cause of patient dissatisfaction. There is little evidence regarding revision arthroplasty in this patient population. The purpose of this study is to evaluate outcomes after revision TKA for arthrofibrosis. METHODS: We retrospectively reviewed 46 consecutive revision TKAs for arthrofibrosis between 2007 and 2015 with minimum 2-year follow-up. Range of motion (ROM), complication rates, and Knee Society Scores (KSS) were recorded. RESULTS: Patients were followed for a mean of 59 months. ROM and KSS significantly improved: with flexion improving from 88° to 103° and extension improving from 11° to 3° (P < .001). There was not a relationship between patient or surgical factors and outcomes in this study. The rate of complications was 28.2% with a 17.4% reoperation rate. CONCLUSION: While revision for arthrofibrosis after TKA can be associated with significant improvements in ROM and KSS, caution is advised given high rates of revisions, reoperations, and complications. Thirty percent of patients in this series had a decrease in one or more component of the KSS or a net decrease in arc of motion after revision surgery.

Radiographic Changes in Nonoperative Contralateral Knee After Unilateral Total Knee Arthroplasty.

BACKGROUND: Some patients perceive symptomatic improvement in the contralateral knee after unilateral total knee arthroplasty (TKA). This so-called "splinting effect" has been observed but has not been radiographically evaluated. METHODS: A retrospective review of patients with bilateral knee osteoarthritis treated with unilateral TKA was performed. Patients were subcategorized into 2 groups based on whether contralateral TKA was performed within the 2-year period. Contralateral radiographic measurements were performed. RESULTS: Forty-four of 203 patients had contralateral TKA performed within 2 years. Preoperative parameters were significantly worse in the bilateral group. By 6 weeks postoperatively, mechanical axis plumbline improved approximately 1°, with more change in those patients with preoperative varus alignment. Larger delta changes were also present in bilateral group preoperative to 1-year radiographs for tibiofemoral angle and joint space widening. CONCLUSION: In patients with bilateral knee osteoarthritis who undergo unilateral TKA, a splinting effect may be present and measurable radiographically with improvement of contralateral mechanical axis plumbline. Further research is necessary to determine whether this improvement may delay contralateral TKA implantation.

Medial Tibial Stress Shielding: A Limitation of Cobalt Chromium Tibial Baseplates.

BACKGROUND: Stress shielding is a well-recognized complication associated with total knee arthroplasty. However, this phenomenon has not been thoroughly described. Specifically, no study to our knowledge has evaluated the radiographic impact of utilizing various tibial component compositions on tibial stress shielding. METHODS: We retrospectively reviewed 3 cohorts of 50 patients that had a preoperative varus deformity and were implanted with a titanium, cobalt chromium (CoCr), or an all polyethylene tibial implant. A radiographic comparative analysis was performed to evaluate the amount of medial tibial bone loss in each cohort. In addition, a clinical outcomes analysis was performed on the 3 cohorts. RESULTS: The CoCr was noted to have a statistically significant increase in medial tibial bone loss compared with the other 2 cohorts. The all polyethylene cohort had a statistically significantly higher final Knee Society Score and was associated with the least amount of stress shielding. CONCLUSION: The CoCr tray is the most rigid of 3 implants that were compared in this study. Interestingly, this cohort had the highest amount of medial tibial bone loss. In addition, 1 patient in the CoCr cohort had medial soft tissue irritation which was attributed to a prominent medial tibial tray which required revision surgery to mitigate the symptoms.

Femoral Implant Design Modification Decreases the Incidence of Patellar Crepitus in Total Knee Arthroplasty.

BACKGROUND: Patellar crepitus is a complication most commonly seen in patients implanted with a posterior-stabilized total knee arthroplasty (TKA). Recently, design changes in the patellofemoral geometry and the intercondylar box ratio have been optimized in newer TKA designs. A comparative study was performed to analyze the incidence of patellar crepitus between a historical vs modern TKA design. METHODS: A retrospective review of all patients at our institution that underwent a primary TKA with either a PFC Sigma or Attune posterior-stabilized TKA (DePuy, Inc, Warsaw, IN), with a minimum of 1-year follow-up duration was performed. A total of 1165 participants implanted with the PFC Sigma and 728 with the Attune design were analyzed. Patellar crepitus incidence, functional scores, and range of motion were recorded at each follow-up appointment. Statistical analyses were performed between the 2 groups to determine if there were differences in clinical outcomes. RESULTS: The incidence of crepitus in participants implanted with the Attune was 0.55% vs 6.26% in the PFC Sigma cohort (P < .001) at 1 year vs. 0.83% vs 9.4%, respectively at 2 years post operatively (P < .001). There were small differences in extension, flexion, and Knee Society Scores between the 2 groups that were not clinically meaningful. CONCLUSION: The Attune posterior-stabilized TKA demonstrated substantially less patellofemoral crepitus incidence than the historical control. We hypothesize that these findings are related to femoral component changes including a thinner and narrower anterior flange and a reduced femoral intercondylar box ratio.

Porous-Coated Metaphyseal Sleeves for Severe Femoral and Tibial Bone Loss in Revision TKA.

BACKGROUND: Metaphyseal bone loss is commonly encountered in revision total knee arthroplasty (TKA). Anderson Orthopaedic Research Institute types 2 and 3 defects generally require some form of metaphyseal fixation or augmentation. This study evaluates the midterm results of stepped, porous-coated metaphyseal sleeves in revision TKA in the setting of severe bone loss. METHODS: Patients who underwent revision TKA using metaphyseal sleeves from March 2006 to May 2014 at our institution were identified from a prospective research database. Preoperative patient characteristics and operative data were reviewed. Postoperative outcomes were compared with preoperative values. Primary study outcomes included complications, reoperations, radiographic assessment of sleeve osteointegration, and survivorship. RESULTS: One hundred sixteen knees (108 patients) underwent revision TKA with 152 metaphyseal sleeves (111 tibial and 41 femoral). Anderson Orthopaedic Research Institute defect classification included 5 type 2A, 89 type 2B, and 17 type 3 tibial defects; and 3 type 2A, 34 type 2B, and 4 type 3 femoral defects. There were 3 intraoperative fractures (1.9%) associated with sleeve preparation and/or insertion. Six knees (5 patients) were lost to follow-up and 5 patients (6 knees) died before 2 years. Of the remaining 104 knees (98 patients, 134 sleeves), mean follow-up was 5.3 years (range 2-9.6 years). Nineteen knees (16.4%) required reoperation, most commonly for recurrent infection. Only one sleeve demonstrated radiographic evidence of failed osteointegration, but did not require revision. Two sleeves (1.5%) required removal and/or resection for recurrent infection. CONCLUSION: This large retrospective series illustrates the utility of porous metaphyseal sleeves in revision TKA with a low rate of intraoperative complications, excellent osteointegration, and long-term fixation.

What Preoperative Radiographic Parameters Are Associated With Increased Medial Release in Total Knee Arthroplasty?

BACKGROUND: Preoperative varus deformity of the knee is a common malalignment in patients undergoing primary total knee arthroplasty (TKA). We are unaware of any studies that have correlated how various preoperative radiographic parameters can predict the amount of medial releases performed to achieve optimal coronal alignment and ligamentous balance. METHODS: A retrospective review was performed on 67 patients who required at least a medial tibial reduction osteotomy (MTRO) during primary TKA to achieve coronal balance. This patient population was matched 1:1 to another cohort of TKA patients by age, gender, and body mass index who did not require an MTRO. A radiographic evaluation was used to compare the 2 cohorts. RESULTS: Preoperatively, the MTRO cohort was noted to have significantly increased varus tibiofemoral (86.12° vs 93.43°), tibial articular surface (85.79° vs 87.54°), and medial tibial articular surface angles (75.22° vs 85.34°) compared to the control cohort. The MTRO cohort had 3.13 mm of medial tibial offset and 9.06 mm of lateral joint space opening and the control cohort had 0.09 mm and 4.07 mm, respectively. The medial tibial articular surface angle and lateral joint space widening were statistically associated with the MTRO cohort. The final tibiofemoral angle in the MTRO cohort was 92.43° and was 93.40° in the control cohort. CONCLUSION: The MTRO cohort was noted to have several preoperative radiographic parameters that were significantly different than the control cohort. However, the medial tibial articular surface angle and lateral joint space widening were the only radiographic parameters that were statistically associated with requiring an MTRO.

Tibial Tray Thickness Significantly Increases Medial Tibial Bone Resorption in Cobalt-Chromium Total Knee Arthroplasty Implants.

BACKGROUND: Stress shielding is an uncommon complication associated with primary total knee arthroplasty. Patients are frequently identified radiographically with minimal clinical symptoms. Very few studies have evaluated risk factors for postoperative medial tibial bone loss. We hypothesized that thicker cobalt-chromium tibial trays are associated with increased bone loss. METHODS: We performed a retrospective review of 100 posterior stabilized, fixed-bearing total knee arthroplasty where 50 patients had a 4-mm-thick tibial tray (thick tray cohort) and 50 patients had a 2.7-mm-thick tibial tray (thin tray cohort). A clinical evaluation and a radiographic assessment of medial tibial bone loss were performed on both cohorts at a minimum of 2 years postoperatively. RESULTS: Mean medial tibial bone loss was significantly higher in the thick tray cohort (1.07 vs 0.16 mm; P = .0001). In addition, there were significantly more patients with medial tibial bone loss in the thick tray group compared with the thin tray group (44% vs 10%, P = .0002). Despite these differences, there were no statistically significant differences in range of motion, knee society score, complications, or revision surgeries performed. CONCLUSION: A thicker cobalt-chromium tray was associated with significantly more medial tibial bone loss. Despite these radiographic findings, we found no discernable differences in clinical outcomes in our patient cohort. Further study and longer follow-up are needed to understand the effects and clinical significance of medial tibial bone loss.

Concentration-dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

Mechanisms of nuclear size regulation in model systems and cancer.

Changes in nuclear size have long been used by cytopathologists as an important parameter to diagnose, stage, and prognose many cancers. Mechanisms underlying these changes and functional links between nuclear size and malignancy are largely unknown. Understanding mechanisms of nuclear size regulation and the physiological significance of proper nuclear size control will inform the interplay between altered nuclear size and oncogenesis. In this chapter we review what is known about molecular mechanisms of nuclear size control based on research in model experimental systems including yeast, Xenopus, Tetrahymena, Drosophila, plants, mice, and mammalian cell culture. We discuss how nuclear size is influenced by DNA ploidy, nuclear structural components, cytoplasmic factors and nucleocytoplasmic transport, the cytoskeleton, and the extracellular matrix. Based on these mechanistic insights, we speculate about how nuclear size might impact cell physiology and whether altered nuclear size could contribute to cancer development and progression. We end with some outstanding questions about mechanisms and functions of nuclear size regulation.

Protein kinase C activity modulates nuclear Lamin A/C dynamics in HeLa cells.

The nuclear lamina serves important functions in the nucleus, providing structural support to the nuclear envelope and contributing to chromatin organization. The primary proteins that constitute the lamina are nuclear lamins whose functions are impacted by post-translational modifications, including phosphorylation by protein kinase C (PKC). While PKC-mediated lamin phosphorylation is important for nuclear envelope breakdown during mitosis, less is known about interphase roles for PKC in regulating nuclear structure. Here we show that overexpression of PKC ß, but not PKC α, increases the Lamin A/C mobile fraction in the nuclear envelope in HeLa cells without changing the overall structure of Lamin A/C and Lamin B1 within the nuclear lamina. Conversely, knockdown of PKC ß, but not PKC α, reduces the Lamin A/C mobile fraction. Thus, we demonstrate an isoform-specific role for PKC in regulating interphase Lamin A/C dynamics outside of mitosis.

Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers.

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.

Epidermal cell fusion promotes the transition from an embryonic to a larval transcriptome in C. elegans.

Cell fusion is a fundamental process in the development of multicellular organisms, yet its impact on gene regulation, particularly during crucial developmental stages, remains poorly understood. The Caenorhabditis elegans epidermis comprises 8-10 syncytial cells, with the largest integrating 139 individual nuclei through cell-cell fusion governed by the fusogenic protein EFF-1. To explore the effects of cell fusion on developmental progression and associated gene expression changes, we conducted transcriptomic analyses of eff-1 fusion-deficient mutants. Our RNAseq findings showed widespread transcriptomic changes that were enriched for epidermal genes and key molecular pathways involved in epidermal function during larval development. Subsequent single-molecule fluorescence in situ hybridization validated the altered expression of mRNA transcripts, confirming quantifiable changes in gene expression in the absence of embryonic epidermal fusion. These results underscore the significance of cell-cell fusion in shaping transcriptional programs during development and raise questions regarding the precise identities and specialized functions of different subclasses of nuclei within developing syncytial cells and tissues.