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Background: Patients with hematologic malignancy (HM) are hypothesized to be at high risk of poor outcomes with coronavirus disease 2019 (COVID-19), due to disease and therapy-related immunosuppression. Despite this, there are minimal reported data describing the outcomes of HM patients with COVID-19. Methods: The COVID-19 and Cancer Consortium (CCC19) (NCT04354701) is an international registry aimed at investigating the clinical course and complications of COVID-19 in patients with cancer. The CCC19 cohort includes patients with active cancer or a history of cancer with presumed or laboratory-confirmed COVID-19. The registry contains de-identified patient demographics, information regarding cancer diagnosis and treatment history, COVID-19 treatments, and clinical outcomes. Patients greater than 18 years of age with laboratory-confirmed, symptomatic COVID-19 and HM diagnoses were included in this study. The primary outcome is a composite of severe COVID-19 illness (composed of mechanical ventilation, severe illness requiring hospitalization, intensive care unit (ICU) requirement, or death); mechanical ventilation, ICU level of care, supplemental oxygen, and 30-day mortality are reported as secondary outcomes. Baseline characteristics are reported for the entire cohort. Reported clinical outcomes are stratified by cancer type, cancer status, line of therapy received (never treated, first, second or later), receipt of cellular therapy or transplant (none, within 12 months, >12 months prior to COVID-19 diagnosis), last receipt of cytotoxic therapy (within 4 weeks, 1-3 months, 3-12 months prior to COVID-19 diagnosis), and receipt of HM therapies under investigation as repurposed treatments for COVID-19 (Bruton tyrosine kinase (BTK) inhibitors, Janus kinase (JAK) inhibitors, Bcr-Abl kinase inhibitors). Results: From March 17, 2020 to July 31, 2020, a total of 757 patients with HM and COVID-19 were enrolled and met inclusion criteria. Median follow-up was 30 days (IQR 17-70 days). Median age was 65 years (IQR 55-75), 62% (470) were over age 60, 57% were men, 45% were non-Hispanic white (22% Black, 18% Hispanic, 5% other), 39% were former or current smokers, 27% were obese, 18% had Eastern Cooperative Oncology Group (ECOG) performance status ≥2, and 51% were on active treatment within 3 months of COVID-19 diagnosis. Among patients with HM, 281 (37%) developed the primary endpoint. Five-hundred and eleven patients (67%) were hospitalized (some with non-severe disease), 188 (25%) required ICU level of care, 133 (18%) required mechanical ventilation, 409 (54%) required supplemental oxygen, and 143 (19%) died within 30 days of COVID-19 diagnosis. Stratified rates of severe outcomes are shown in the Table. The rate of severe COVID-19 was highest in patients with chronic lymphocytic leukemia (53%), and lowest in patients with Hodgkin lymphoma (23%). Patients receiving cytotoxic systemic therapy within 3 months of COVID-19 diagnosis had higher rates of severe COVID-19 (41%) and 30-day mortality (28%) than patients who completed treatment 3-12 months (26% severe COVID-19, 16% 30-day mortality) or more than 12 months (29% severe COVID-19, 13% 30-day mortality) prior to COVID-19 diagnosis. Patients receiving cellular therapy or transplant within a year prior to COVID-19 diagnosis had similar rates of severe COVID-19 (36% v. 38%) and 30-day mortality (23% v. 19%) to patients who had not received such therapies within a year prior to COVID-19 diagnosis. Patients on second-line or later therapy experienced similar rates of poor outcomes (42% severe COVID-19, 20% 30-day mortality) to patients on first-line therapy (39% severe COVID-19, 18% 30-day mortality) and untreated patients (42% severe COVID-19, 20% 30-day mortality). Outcomes for patients receiving therapies under investigation as repurposed COVID-19 treatments were similar to the cohort at large. Conclusions: This is the largest cohort study to date describing COVID-19 outcomes in patients with HM. Rates of severe COVID-19 outcomes including death were high. Unadjusted rates of severe COVID-19 outcomes were higher in patients with previously described risk factors such as advanced age, poor performance status, and progressive disease, as well as those receiving recent cytotoxic therapy. Outcomes varied widely by malignancy but were similar across treatment contexts. Additional data collection and analyses are ongoing. Disclosures Lynch: Bayer: Research Funding; Rhizen Pharmaceuticals: Research Funding; Incyte: Research Funding; TG Therapeutics: Research Funding; Takeda: Research Funding; Juno Therpeutics: Research Funding; Genentech: Research Funding; MorphoSys: Consultancy; Cyteir: Research Funding. Bakouny:BMS: Research Funding; Genentech: Research Funding. Bhutani:Sanofi: Consultancy, Research Funding. Shah:American Cancer Society and the Hope Foundation for Cancer Research: Research Funding; National Cancer Institute: Research Funding. Lyman:Mylan: Consultancy; Beyond Spring: Consultancy; Samsung: Consultancy; Sandoz: Consultancy; Invitae: Consultancy; Spectrum: Consultancy; G1 Therapeutics: Consultancy; Amgen: Research Funding. Kuderer:Janssen: Consultancy; G1 Therapeutics: Consultancy; Beyond Springs: Consultancy; Spectrum Pharmaceuticals: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Consultancy; celldex: Consultancy; Total Health: Consultancy; Invitae: Consultancy. Warner:HemOnc.orgLLC: Other: Shareholder/Stockholder/Stock options; National Cancer Institute: Research Funding; IBM Watson Health: Consultancy; Westat: Consultancy. Mesa:Bristol Myers Squibb: Research Funding; Incyte: Research Funding; AbbVie: Research Funding; Samus Therapeutics: Research Funding; Genentech: Research Funding; Promedior: Research Funding; CTI BioPharma: Research Funding; Novartis: Consultancy; Sierra Oncology: Consultancy; LaJolla Pharmaceutical Company: Consultancy. Thompson:AIM Specialty Health, BMS, GlaxoSmithKline, Takeda, Via Oncology: Membership on an entity's Board of Directors or advisory committees; Doximity: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Synapse Precision Medical Council: Other: Travel expenses.

When the COVID-19 pandemic began, formal frameworks to collect data about affected patients were lacking. The COVID-19 and Cancer Consortium (CCC19) was formed to collect granular data on patients with cancer and COVID-19 at scale and as rapidly as possible. CCC19 has grown from five initial institutions to 125 institutions with >400 collaborators. More than 5,000 cases with complete baseline data have been accrued. Future directions include increased electronic health record integration for direct data ingestion, expansion to additional domestic and international sites, more intentional patient involvement, and granular analyses of still-unanswered questions related to cancer subtypes and treatments.

BACKGROUND: E4402 was a randomized phase III study comparing two different rituximab dosing strategies for patients with previously untreated, low tumor burden follicular lymphoma (FL). The primary endpoint was time to treatment failure. The initial publication (Kahl, JCO 2014) demonstrated that a retreatment strategy utilized less drug and produced comparable time to treatment failure compared to a maintenance strategy. Here we provide long term follow up results, focusing on response duration, time to first cytotoxic therapy, overall survival, and risk of histologic transformation. METHODS: Eligible patients had untreated, low tumor burden (GELF criteria) FL. Patients received R 375 mg/m 2 weekly x 4 and responders were randomized to maintenance rituximab (MR) (single dose R q 3 months) or retreatment rituximab (RR) (R weekly x 4 doses at disease progression). Each strategy was continued until treatment failure. The primary endpoint, time to treatment failure, was defined as progression within 6 months of last R, no response to R retreatment, initiation of alternative therapy, or inability to complete protocol therapy. Secondary endpoints included time to first cytotoxic therapy, quality of life and safety. The ECOG Data Monitoring Committee recommended release of study results at a planned interim analysis in September 2011 and patients and providers were notified of results. Time to treatment failure data collection halted with release of the results but limited data collection on time to first cytotoxic therapy, response duration, and risk of histologic transformation continued. INITITIAL RESULTS: From 11/03 to 9/08, 384 patients with FL were enrolled. Complete or partial response was achieved in 289 patients (71%), who were then randomized to MR (n=146) or RR (n=143). Demographic features were similar in the two arms: median age 59 years; ECOG PS 0-1 in all patients, and FLIPI low-risk (14.9 vs. 16.4%), intermediate-risk (46.3 vs. 42.9%) and high-risk (38.8 vs. 40.7%) for MR vs. RR, respectively. At initial publication, with a median follow-up of 3.8 years, the time to treatment failure was 3.9 years for MR vs. 3.6 years for RR (p=NS). LONG TERM FOLLOW UP RESULTS: Immunoglobulin levels and risks for serious infections/late complications in MR patients will be updated at the annual meeting. For the endpoint of time to first cytotoxic therapy, the median follow up is 8.7 years. At 7 years, 83% of MR and 63% of RR remained free from first cytotoxic therapy (HR 2.37; 95% CI 1.50 - 3.76) [Figure 1]. For the endpoint of response duration, the median follow up is 12.1 years. At 10 years, 66% of the MR patients remained in their 1 st remission compared to 30% of the RR patients who remained in their 1 st remission [Figure 2]. The median response duration for RR patients receiving a single 4-week course of rituximab was 3.25 years. There was no difference in the overall survival at 10 years, 84% for MR vs. 83% for RR. There was a trend towards a lower risk of histologic transformation for patients receiving MR (n = 4) compared to RR (n = 11) (p = 0.11). CONCLUSIONS: With long term follow up, the RESORT data indicates that in previously untreated, low tumor burden, follicular lymphoma, MR was superior to RR for delaying time to first cytotoxic therapy and for response duration, with a trend towards reducing the risk of histologic transformation. MR did not improve the overall survival. The original publication concluded that the time to treatment failure is similar between the two dosing strategies. Due to study design, time to treatment failure could not be analyzed in this long term follow up analysis. Compared to the historical benchmark of 3 years median time to first cytotoxic therapy when watch and wait is utilized, single agent rituximab, administered by either dosing strategy, was highly effective at delaying the time to first cytotoxic therapy. Figure 1 Figure 1. Disclosures Kahl: AbbVie, Adaptive, ADCT, AstraZeneca, Bayer, BeiGene, Bristol-Myers Squibb, Celgene, Genentech, Incyte, Janssen, Karyopharm, Kite, MEI, Pharmacyclics, Roche, TG Therapeutics, and Teva: Consultancy; AbbVie, Acerta, ADCT, AstraZeneca, BeiGene, Genentech: Research Funding. Habermann: Incyte: Other: Scientific Advisory Board; Tess Therapeutics: Other: Data Monitoring Committee; Seagen: Other: Data Monitoring Committee; Morphosys: Other: Scientific Advisory Board; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Schuster: Abbvie: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Adaptive Biotechnologies: Research Funding; BeiGene: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; DTRM: Research Funding; Genetech: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Incyte: Research Funding; Juno Theraputics: Consultancy, Research Funding; Loxo Oncology: Consultancy; Merck: Research Funding; Nordic Nanovector: Consultancy; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; Pharmaclcyclics: Research Funding; Tessa Theraputics: Consultancy; TG Theraputics: Research Funding. Fenske: TG Therapeutics: Consultancy, Speakers Bureau; Servier Pharmaceuticals: Consultancy; Seattle Genetics: Speakers Bureau; Sanofi: Speakers Bureau; Pharmacyclics: Consultancy; MorphoSys: Consultancy; Kite (Gilead): Speakers Bureau; KaryoPharm: Consultancy; CSL Therapeutics: Consultancy; Bristol-Myers Squibb: Speakers Bureau; Biogen: Consultancy; Beigene: Consultancy; AstraZeneca: Speakers Bureau; ADC Therapeutics: Consultancy; Adaptive Biotechnologies: Consultancy; AbbVie: Consultancy.

Asymmetric inheritance of cell organelles determines the fate of daughter cells. Böckler et al. use yeast as a model to demonstrate that the dynamics of mitochondrial fusion, fission, and transport determine partitioning of mitochondria and cytosolic protein aggregates, which is critical for rejuvenation of daughter cells., Partitioning of cell organelles and cytoplasmic components determines the fate of daughter cells upon asymmetric division. We studied the role of mitochondria in this process using budding yeast as a model. Anterograde mitochondrial transport is mediated by the myosin motor, Myo2. A genetic screen revealed an unexpected interaction of MYO2 and genes required for mitochondrial fusion. Genetic analyses, live-cell microscopy, and simulations in silico showed that fused mitochondria become critical for inheritance and transport across the bud neck in myo2 mutants. Similarly, fused mitochondria are essential for retention in the mother when bud-directed transport is enforced. Inheritance of a less than critical mitochondrial quantity causes a severe decline of replicative life span of daughter cells. Myo2-dependent mitochondrial distribution also is critical for the capture of heat stress–induced cytosolic protein aggregates and their retention in the mother cell. Together, these data suggest that coordination of mitochondrial transport, fusion, and fission is critical for asymmetric division and rejuvenation of daughter cells.

The shape of kinetoplastids, such as Trypanosoma brucei, is precisely defined during the stages of the life cycle and governed by a stable subpellicular microtubule cytoskeleton. During the cell cycle and transitions between life cycle stages this stability has to transiently give way to a dynamic behaviour to enable cell division and morphological rearrangements. How these opposing requirements of the cytoskeleton are regulated is poorly understood. Two possible levels of regulation are activities of cytoskeleton-associated proteins and microtubule posttranslational modifications (PTMs). Here, we investigate the functions of two putative tubulin polyglutamylases in T. brucei, TTLL6A and TTLL12B. Depletion of both proteins leads to a reduction in tubulin polyglutamylation in situ and is associated with disintegration of the posterior cell pole, loss of the microtubule plus end-binding protein EB1 and alterations of microtubule dynamics. We also observe a reduced polyglutamylation of the flagellar axoneme. Quantitative motility analysis reveals that the PTM disbalance correlates with a transition from directional to diffusive cell movement. These data show, that microtubule polyglutamylation has an important role in regulating cytoskeletal architecture and motility in this parasite.

Major steps in embryonic development, e.g. body axes formation or asymmetric cell divisions, rely on symmetry-breaking events and gradient formation. Using three-dimensional time-resolved lightsheet microscopy, we have studied a prototypical example for self-organized gradient formation in the model organism Caenorhabditis elegans. In particular, we have monitored in detail the formation of a concentration and mobility gradient of PIE-1 proteins as well as the partitioning behavior of vital organelles prior to the first, asymmetric cell division. Our data confirm the emergence of a concentration gradient of PIE-1 along the embryo’s anterior–posterior (AP) axis but they also reveal a previously unseen depletion zone near to the cell cortex that is not present for MEX-5 proteins. Time-resolved spatial diffusion maps, acquired with SPIM-FCS, highlight the successive emergence of a mobility gradient of PIE-1 along the AP axis and suggest an almost linear decrease of fast diffusing PIE-1 proteins along the AP axis. Quantifying the subordinated dissemination of vital organelles prior to the first cell division, i.e. gradient formation on larger length scales, we find a significant asymmetry in the partitioning of the endoplasmic reticulum and mitochondria to the anterior and posterior part of the cell, respectively. Altogether, our spatiotemporally resolved data indicate that current one-dimensional model descriptions for gradient formation during C. elegans embryogenesis, i.e. a mere projection to the AP axis, might need an extension to a full three-dimensional description. Our data also advocate the use of lightsheet microscopy techniques to capture the actual three-dimensional nature of embryonic self-organization events.

The trans-Golgi network (TGN) is the main secretory pathway sorting station, where cargoes are packed into appropriate transport vesicles targeted to specific destinations. Exomer is a cargo adaptor necessary for direct transport of a subset of cargoes from the TGN to the plasma membrane in yeast. Here, we show that unlike classical adaptor complexes, exomer is not recruited en bloc to the TGN, but rather assembles through a stepwise pathway, in which first the scaffold protein Chs5 and then the cargo-binding units, the ChAPs, are recruited. Although all ChAPs are able to assemble functional exomer complexes, they do so with different efficiencies. The mutual relationship between ChAPs varies from cooperation to competition depending on their expression levels and affinities to Chs5 allowing an optimized and efficient cargo transport. The multifactorial assembly pathway results in an exquisitely fine-tuned adaptor complex, enabling the cell to quickly respond and adapt to changes such as stress.

This phase 3 trial (Eastern Cooperative Oncology Group [ECOG] E1A06) compared melphalan, prednisone, and thalidomide (MPT-T) with melphalan, prednisone, and lenalidomide (mPR-R) in patients with untreated multiple myeloma (MM). A noninferiority design was used, and inferiority was defined as a progression-free survival (PFS) hazard ratio (HR) of MPT-T/mPR-R ≤0.82. A total of 306 patients enrolled, with a median age of 75.7 years. Median follow-up was 40.7 months. Median time on therapy was 12.1 months and 23.1 months for the 46.6% of treated patients who received maintenance, with no differences by arm. Median PFS was 21 months on MPT-T and 18.7 months on mPR-R (HR, 0.84; 95% confidence interval, 0.64-1.09). Overall survival was 52.6 months (MPT-T) vs 47.7 months (mPR-R) (P = .476). Per-protocol response rates were 63.6% (MPT-T) and 59.9% (mPR-R) (P = .557). Grade ≥3 nonhematologic toxicity was 59.5% for MPT-T vs 40.0% for mPR-R (P = .001). Second malignancies were observed in 18 MPT-T patients vs 14 mPR-R patients. Quality-of-life analysis favored mPR-R by induction end (P = .007). Use of MPT-T or mPR-R in elderly patients with untreated MM demonstrates no statistical or clinically relevant differences in response rates, PFS, and OS; however, quality of life at end of induction was improved and lower toxicity reported with mPR-R. This trial was registered at www.clinicaltrials.gov as #NCT00602641.

A microtubule-independent membranous “spindle envelope” confines spindle assembly and accounts for faithful chromosome segregation., 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.

Reconstitution of cellular organelles in vitro offers the possibility to perform quantitative and qualitative experiments in a controlled environment that cannot be done with the same accuracy in living cells. Following a previous report, the subsequent list of protocols describes how to reconstitute and quantify a tubular ER network in vitro based on purified microsomes from culture cells and cytosol from Xenopus laevis egg extracts. Biological material preparation and reconstitution assays require mostly basic laboratory instrumentation and chemicals, and can be executed without any specific training, making them appealing to a wide range of laboratories. Moreover, to promote conditions that are markedly more reflective of in vivo environments, this method describes for the first time in the literature, the purification of microsomes from HeLa cells in some detail. Basic Protocol 1 in this article describes the reconstitution process on different substrates including the associated fluorescence imaging process. Purification of ER microsomes and cytosol, both of which are needed for this approach, are described in detail in Support Protocols 1 and 2, respectively. Coating of surfaces with polyacrylamide gels is described in Support Protocol 3. Basic Protocol 2 outlines how to segment and skeletonize fluorescence images of ER networks, and how to quantify segment lengths between the network's branching points. The described quantitative evaluation provides a meaningful approach to analyze the topology and geometry of organelle structures. © 2017 by John Wiley & Sons, Inc.

Asymmetric cell divisions are of fundamental importance for developmental processes, e.g. for the generation of founder cells. Prime examples are asymmetric cell divisions in the P lineage during early embryogenesis of the model organismCaenorhabditis elegans. However, due to a lack of quantitative data it has remained unclear how frequent unequal daughter cell sizes emerge in the nematode’s early embryogenesis, and whether these originate from sterical or biochemical cues. Using quantitative light-sheet microscopy, we have found that about 40% of all cell divisions inC. elegansuntil gastrulation generate daughter cells with significantly different volumes. Removing the embryo’s rigid eggshell revealed asymmetric divisions in somatic cells to be primarily induced by steric effects. Division asymmetries in the germline remained unaltered and were correctly reproduced by a model based on a cell-size independent, eccentric displacement of the metaphase plate. Our data suggest asymmetric cell divisions to be essential for establishing important cell-cell interactions that eventually fuel a successful embryogenesis.Summary statementAbout 40% of all cell divisions in earlyC. elegansembryogenesis are found to be asymmetric. A cell-size independent displacement of the mitotic spindle explains division asymmetries in the germline whereas the confining eggshell induces asymmetries of somatic cells.

Summary Character and rapidity of protein diffusion in intracellular fluids are key determinants of the dynamics and steady state of a plethora of biochemical reactions [1, 2]. So far, an anomalous diffusion in cytoplasmic fluids with viscoelastic and even glassy characteristics has been reported in a variety of organisms on several length scales and timescales [3–6]. Here, we show that the contiguous fluid of former cytoplasm and nucleoplasm features an anisotropically varying diffusion of macromolecules during eukaryotic cell division. In metaphase, diffusion in the contiguous nucleocytoplasmic fluid appears less anomalous along the spindle axis as compared to perpendicular directions. As a consequence, the long-time diffusion of macromolecules preferentially points along the spindle axis, leading to prolonged residence of macromolecules in the spindle region. Based on our experimental data, we suggest that anisotropic diffusion facilitates the encounter and interaction of spindle-associated proteins, e.g., during the formation of a dynamic spindle matrix [7].

Biochemical signaling pathways in developmental processes have been extensively studied, yet the role of mechanical cues during embryogenesis is much less explored. Here we have used selective plane illumination microscopy in combination with a simple mechanical model to quantify and rationalize cell motion during early embryogenesis of the small nematode Caenorhabditis elegans. As a result, we find that cell organization in the embryo until gastrulation is well described by a purely mechanical model that predicts cells to assume positions in which they face the least repulsive interactions from other cells and the embryo’s egg shell. Our findings therefore suggest that mechanical interactions are key for a rapid and robust cellular arrangement during early embryogenesis of C. elegans.

The embryogenesis of the small nematode Caenorhabditis elegans is a remarkably robust self-organization phenomenon. Cell migration trajectories in the early embryo, for example, are well explained by mechanical cues that push cells into positions where they experience the least repulsive forces. Yet, how this mechanically guided progress in development is properly timed has remained elusive so far. Here, we show that cell volumes and division times are strongly anticorrelated during the early embryogenesis of C. elegans with significant differences between somatic cells and precursors of the germline. Our experimental findings are explained by a simple model that in conjunction with mechanical guidance can account for the fail-safe early embryogenesis of C. elegans.

Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1μm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion.

Sorting of membrane proteins in eukaryotic cells is a complex yet vital task that involves several 10,000 molecular players. Sorting takes place not only along the early secretory pathway, i.e., between the endoplasmic reticulum and the Golgi apparatus, but also between other organelles, including exchange with the cell's plasma membrane. Traditionally, specific binary interactions between proteins have been made responsible for most of the protein sorting. A more active role of lipids, however, became visible in recent years. Not only do lipids in complex membranes show domain formation that may support/suppress sorting events, but also collective, membrane-mediated interactions have emerged as a robust physico-chemical mechanism to drive protein sorting. Here, we will review recent insights into these aspects.

p24 proteins are required for secretion of Wnt ligands Wnt proteins are signalling molecules that follow a dedicated secretory pathway. The authors here identify novel components of this pathway, providing evidence that p24 proteins act as cargo receptors in an anterograde secretory route for Wnts., During development and disease, the exocytosis of signalling molecules, such as Wnt ligands, is essential to orchestrate cellular programs in multicellular organisms. However, it remains a largely unresolved question whether signalling molecules follow specialized transport routes through the exocytic pathway. Here we identify several Drosophila p24 proteins that are required for Wnt signalling. We demonstrate that one of these p24 proteins, namely Opossum, shuttles in the early secretory pathway, and that the Drosophila Wnt proteins are retained in the absence of p24 proteins. Our results indicate that Wnt secretion relies on a specialized anterograde secretion route with p24 proteins functioning as conserved cargo receptors.

A multitude of transmembrane proteins enters the endoplasmic reticulum (ER) as unfolded polypeptide chains. During their folding process, they interact repetitively with the ER's quality control machinery. Here, we have used fluorescence correlation spectroscopy to probe these interactions for a prototypical transmembrane protein, VSVG ts045, in vivo. While both folded and unfolded VSVG ts045 showed anomalous diffusion, the unfolded protein had a significantly stronger anomaly. This difference subsided when unfolded VSVG ts045 was in a complex with its chaperone calnexin, or when a mutant form of VSVG ts045 with only one glycan was used. Our experimental data and accompanying simulations suggest that the folding sensor of the quality control (UGT1) oligomerizes unfolded VSVG ts045, leading to a more anomalous/obstructed diffusion. In contrast, calnexin dissolves the oligomers, rendering unfolded VSVG ts045 more mobile, and hence prevents poisoning of the ER.

TIF-IA is a basal transcription factor of RNA polymerase I (Pol I) that is a major target of the JNK2 signaling pathway in response to ribotoxic stress. Using advanced fluorescence microscopy and kinetic modeling we elucidated the subcellular localization of TIF-IA and its exchange dynamics between the nucleolus, nucleoplasm and cytoplasm upon ribotoxic stress. In steady state, the majority of (GFP-tagged) TIF-IA was in the cytoplasm and the nucleus, a minor portion (7%) localizing to the nucleoli. We observed a rapid shuttling of GFP-TIF-IA between the different cellular compartments with a mean residence time of approximately 130 s in the nucleus and only approximately 30 s in the nucleoli. The import rate from the cytoplasm to the nucleus was approximately 3-fold larger than the export rate, suggesting an importin/exportin-mediated transport rather than a passive diffusion. Upon ribotoxic stress, GFP-TIF-IA was released from the nucleoli with a half-time of approximately 24 min. Oxidative stress and inhibition of protein synthesis led to a relocation of GFP-TIF-IA with slower kinetics while osmotic stress had no effect. The observed relocation was much slower than the nucleo-cytoplasmic and nucleus-nucleolus exchange rates of GFP-TIF-IA, indicating a time-limiting step upstream of the JNK2 pathway. In support of this, time-course experiments on the activity of JNK2 revealed the activation of the JNK kinase as the rate-limiting step.

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIalpha (PI4K-IIIalpha) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIalpha, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

Cellular membranes are typically decorated with a plethora of embedded and adsorbed macromolecules, e.g. proteins, that participate in numerous vital processes. With typical surface densities of 30,000 proteins per μm(2) cellular membranes are indeed crowded places that leave only few nanometers of private space for individual proteins. Here, we review recent advances in our understanding of protein crowding in membrane systems. We first give a brief overview on state-of-the-art approaches in experiment and simulation that are frequently used to study crowded membranes. After that, we review how crowding can affect diffusive transport of proteins and lipids in membrane systems. Next, we discuss lipid and protein sorting in crowded membrane systems, including effects like protein cluster formation, phase segregation, and lipid droplet formation. Subsequently, we highlight recent progress in uncovering crowding-induced conformational changes of membranes, e.g. membrane budding and vesicle formation. Finally, we give a short outlook on potential future developments in the field of crowded membrane systems. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Rog.

Alterations in endoplasmic reticulum (ER) cholesterol are fundamental for a variety of cellular processes such as the regulation of lipid homeostasis or efficient protein degradation. We show that reduced levels of cellular sterols cause a delayed ER-to-Golgi transport of the secretory cargo membrane protein ts-O45-G and a relocation to the ER of an endogenous protein cycling between the ER and the Golgi complex. Transport inhibition is characterized by a delay in the accumulation of ts-O45-G in ER-exit sites (ERES) and correlates with a reduced mobility of ts-O45-G within ER membranes. A simple mathematical model describing the kinetics of ER-exit predicts that reduced cargo loading to ERES and not the reduced mobility of ts-O45-G accounts for the delayed ER-exit and arrival at the Golgi. Consistent with this, membrane turnover of the COPII component Sec23p is delayed in sterol-depleted cells. Altogether, our results demonstrate the importance of sterol levels in COPII mediated ER-export.

Summary The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes [1–3]. Both stimulate the GTPase activity of Sar1p [4], which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.

Confocal fluorescence recovery after photobleaching (FRAP) is today the prevalent tool when studying the diffusional and kinetic properties of proteins in living cells. Obtaining quantitative data for diffusion coefficients via FRAP, however, is challenged by the fact that both bleaching and scanning take a finite time. Starting from an experimental case, it is shown by means of computer simulations that this intrinsic temporal limitation can lead to a gross underestimation of diffusion coefficients. Determining the binding kinetics of proteins to membranes with FRAP is further shown to be severely hampered by additional diffusional contributions, e.g. diffusion-limited binding. In some cases, the binding kinetics may even be masked entirely by diffusion. As current efforts to approach biological problems with biophysical models have to rely on experimentally determined model parameters, e.g. binding rates and diffusion constants, it is proposed that the accuracy in evaluating FRAP measurements can be improved by means of accompanying computer simulations.

The random movement of molecules (diffusion) is fundamental to most cellular processes, including enzymatic reactions, signalling, protein-protein interaction, as well as domain and pattern formation. Despite playing a central role, diffusion is, to a large extent, under-appreciated in the cell biology community. One reason for this is that diffusion is rather challenging to study in living cells. This article is intended to explain, at least in part, how we can go about studying diffusion of molecules in living cells, why it is important and how it provides us with important clues about biological systems. As the title 'In a mirror dimly' suggests, we do this by monitoring faint light emitted by fluorescent probes or proteins using advanced optics (e.g. mirrors) and electronics. The data are then fitted and interpreted with mathematical and physical models, providing a glimpse into the world of molecules.

Understanding dynamic processes in cells and multicellular systems requires spatiotemporally resolved imaging techniques with low photobleaching and -toxicity. Following this demand, single-plane illumination microscopes (SPIMs) have recently emerged as powerful tools. Using a custom-made SPIM setup that combines rapid widefield detection with optical sectioning and reduced bleaching, we have performed long-term, three-dimensional in-vivo imaging on early embryos of the small nematode Caenorhabditis elegans [1]. Based on our SPIM data, we were able to quantify the movement and arrangment of cells during early embryogenesis. The remarkable similarity of individual cell trajectories across different embryos is well captured by a simple model that mainly considers mechanical interactions of cells [1].Going beyond mere imaging, we have also implemented a SPIM-based fluorescence correlation spectroscopy (FCS) approach, which allows for quantifying protein diffusion in the early embryo at high spatiotemporal resolution. As a first application, we have determined spatially resolved diffusion maps of the peripheral membrane protein PLC1δ1 in the cytoplasm and on the plasma membrane of worm embryos in the one- and two-cell stage. The obtained results compare favorably to previous reports on the same protein construct. We also have determined time-resolved diffusion maps of the protein PIE-1 which highlight a significantly varying mobility of the protein along the anterior-posterior axis of the embryo before the first, asymmetric cell division. Hence, SPIM-FCS is a well-suited tool for assessing diffusional transport in developmental model organisms.[1] Fickentscher, Struntz & Weiss, Biophys. J. 105, 1805 (2013).

Resident proteins of the exocytic pathway are maintained at various levels through coatomer protein I (COPI)-mediated recycling. Sorting of cargo by COPI requires GTP hydrolysis by ADP-ribosylation factor 1 (ARF-1). This small GTPase recruits coatomer onto Golgi membranes and upon hydrolysis, is thought to release coatomer back into the cytosol. This step requires the activating protein, ARFGAP1. By coupling sorting to a cargo-induced sequestering of ARFGAP1, we have formulated a kinetic proof-reading model that explains how a GTP hydrolysis-driven coat release can yield an active sorting event. The sorting scheme predicts a dependency on the amount of ARFGAP1 and explains the recent experimental findings that ARF-1 and COPI detach with different time constants from the Golgi membrane in vivo.

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd.

Trophoblast stem cells (TSCs) represent the multipotent progenitors that give rise to the different cells of the embryonic portion of the placenta. Here, we analysed the expression of key TSC transcription factorsCdx2,Eomes, andElf5in the early developing placenta of mouse embryos and in cultured TSCs and reveal surprising heterogeneity in protein levels. We analysed persistence of TSCs in the early placenta and find that TSCs remain in the chorionic hinge until E9.5 and are lost shortly afterwards. To define the transcriptional signature of bona fide TSCs, we used inducible gain- and loss-of-function alleles ofEomesorCdx2, andEomesGFP, to manipulate and monitor the core maintenance factors of TSCs, followed by genome-wide expression profiling. Combinatorial analysis of resulting expression profiles allowed for defining novel TSC marker genes that might functionally contribute to the maintenance of the TSC state. Analyses by qRT-PCR andin situhybridisation validated novel TSC- and chorion-specific marker genes, such asBok/Mtd, Cldn26, Duox2, Duoxa2, Nr0b1, andSox21. Thus, these expression data provide a valuable resource for the transcriptional signature of bona fide and early differentiating TSCs and may contribute to an increased understanding of the transcriptional circuitries that maintain and/or establish stemness of TSCs.

The T-box transcription factor Eomes (also known as Tbr2) shows short-lived expression in various localized domains of the embryo, including epiblast cells during gastrulation and intermediate progenitor cells in the cerebral cortex. In these tissues Eomes fulfills crucial roles for lineage specification of progenitors. To directly observe Eomes-dependent cell lineages in the living embryo, we generated a novel dual-fluorescence reporter allele that expresses a membrane-bound tdTomato protein for investigation of cell morphology and a nuclear GFP for cell tracing. This allele recapitulates endogenous EOMES protein expression and is suitable for live imaging. We found that the allele can also be used as a short-to-medium-term lineage tracer, as GFP persists in cells longer than EOMES protein and marks Eomes-dependent lineages with a timeframe of days to weeks depending on the proliferation rate. In summary, we present a novel genetic tool for investigation of Eomes-dependent cell types by live imaging and lineage tracing.

We currently lack a broader mechanistic understanding of the integration of the early secretory pathway with other homeostatic processes such as cell growth. Here, we explore the possibility that Sec16A, a major constituent of endoplasmic reticulum exit sites (ERES), acts as an integrator of growth factor signaling. Surprisingly, we find that Sec16A is a short-lived protein that is regulated by growth factors in a manner dependent on Egr family transcription factors. We hypothesize that Sec16A acts as a central node in a coherent feed-forward loop that detects persistent growth factor stimuli to increase ERES number. Consistent with this notion, Sec16A is also regulated by short-term growth factor treatment that leads to increased turnover of Sec16A at ERES. Finally, we demonstrate that Sec16A depletion reduces proliferation, whereas its overexpression increases proliferation. Together with our finding that growth factors regulate Sec16A levels and its dynamics on ERES, we propose that this protein acts as an integrator linking growth factor signaling and secretion. This provides a mechanistic basis for the previously proposed link between secretion and proliferation.

Summary Cell-fate decisions and pluripotency are dependent on networks of key transcriptional regulators. Recent reports demonstrated additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor TBX3 has been implicated in regulating embryonic stem cell self-renewal and cardiogenesis. Here, we show that TBX3 is dynamically expressed during specification of the mesendoderm lineages in differentiating embryonic stem cells (ESCs) in vitro and in developing mouse and Xenopus embryos in vivo. Forced TBX3 expression in ESCs promotes mesendoderm specification by directly activating key lineage specification factors and indirectly by enhancing paracrine Nodal/Smad2 signaling. TBX3 loss-of-function analyses in the Xenopus underline its requirement for mesendoderm lineage commitment. Moreover, we uncovered a functional redundancy between TBX3 and Tbx2 during Xenopus gastrulation. Taken together, we define further facets of TBX3 actions and map TBX3 as an upstream regulator of the mesendoderm transcriptional program during gastrulation., Graphical Abstract, Highlights • T-box transcription factor TBX3 is involved in early embryonic events • Key transcription factor promoters of mesendoderm formation are occupied by TBX3 • TBX3 promotes mesendodermal fate of mESCs

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Chromosome segregation during mitosis is mediated by the mitotic spindle, a structure composed of microtubules associated with a variety of proteins. Additionally, a distinct biochemical milieu, the 'spindle matrix', has long been proposed to assist this process and recent work provided compelling evidence for its existence in living animal cells. Here we focus on the dynamic properties of such a matrix, highlighting its roles in the spatial and temporal control of mitotic fidelity in respect to recent findings in different systems. Based on these findings and on theoretical principles behind molecular crowding, we challenge the idea of the spindle matrix as one functional entity and propose that some observed phenomena associated with this elusive structure could simply be a consequence of intrinsic cellular architecture and cytoplasmic 'compartmentalization' during mitosis.

Sorting of membrane proteins is of vital importance for living cells. Indeed, roughly one-third of a eukaryotic cell’s proteome consists of peripheral and transmembrane proteins. These need to be properly distributed and dynamically maintained at distinct locations in the compartmentalized cell, and one may wonder how proteins determine where, when, and how to travel to reach a specific organelle. While specific binary interactions between proteins have been invoked in explaining the trafficking and sorting processes, a more active role of lipids in this context has become visible in recent years. In particular, membrane-mediated interactions have been suggested to serve as a robust physicochemical mechanism to facilitate protein sorting. Here, we will review some recent insights into these aspects.

Non-integral membrane proteins frequently act as transduction hubs in vital signaling pathways initiated at the plasma membrane (PM). Their biological activity depends on dynamic interactions with the PM, which are governed by their lateral and cytoplasmic diffusion and membrane binding/unbinding kinetics. Accurate quantification of the multiple kinetic parameters characterizing their membrane interaction dynamics has been challenging. Despite a fair number of approximate fitting functions for analyzing fluorescence recovery after photobleaching (FRAP) data, no approach was able to cope with the full diffusion-exchange problem. Here, we present an exact solution and matlab fitting programs for FRAP with a stationary Gaussian laser beam, allowing simultaneous determination of the membrane (un)binding rates and the diffusion coefficients. To reduce the number of fitting parameters, the cytoplasmic diffusion coefficient is determined separately. Notably, our equations include the dependence of the exchange kinetics on the distribution of the measured protein between the PM and the cytoplasm, enabling the derivation of both k(on) and k(off) without prior assumptions. After validating the fitting function by computer simulations, we confirm the applicability of our approach to live-cell data by monitoring the dynamics of GFP-N-Ras mutants under conditions with different contributions of lateral diffusion and exchange to the FRAP kinetics.

Using coarse-grained membrane simulations we show here that peripheral membrane proteins can form a multitude of higher-order structures due to membrane-mediated interactions. Peripheral membrane proteins characteristically perturb the lipid bilayer in their vicinity which supports the formation of protein assemblies not only within the same but surprisingly also across opposing leaflets of a bilayer. In addition, we also observed the formation of lipid-protein domains on heteregeneous membranes. The clustering ability of proteins, as quantified via the potential of mean force, is enhanced when radius and hydrophobic penetration depth of the proteins increases. Based on our data, we propose that membrane-mediated cluster formation of peripheral proteins supports protein assembly in vivo and hence may play a pivotal role in the formation of templates for signaling cascades and in the emergence of transport intermediates in the secretory pathway., Author Summary Eukaryotic cells are subdivided into a variety of compartments by membranes, i.e. by lipid bilayers into which a multitude of proteins are embedded. About 30% of all protein species in a cell are associated with membranes to perform vital functions, e.g. in signaling and transport pathways. A plethora of membrane-associated proteins, so-called peripheral membrane proteins, penetrate only one monolayer whereas transmembrane proteins span the entire thickness of a lipid bilayer. Despite the crucial role of peripheral membrane proteins in many cellular processes, little is known about the general principles of how they can form higher-order structures that eventually are biologically active units. Using a simulation approach we show and quantify here the influence of membrane-mediated interactions between peripheral membrane proteins. These interactions most likely play a pivotal role in forming higher-order structures in living cells. In particular, template formation in signaling cascades and coat assembly during the formation of transport intermediates may strongly benefit from the phenomena described here.

Sorting of transmembrane proteins is a central task of the secretory pathway. Due to the lack of an organizing mastermind, the decision whether a protein participates in anterograde/retrograde transport or rather stays in its compartment has to be made by a self-organization process on the molecular scale. Minimizing the hydrophobic mismatch between a protein and the surrounding lipid bilayer has been shown to be an important determinant of protein localization. It has remained elusive, however, how mislocalized proteins sense that remote organelles may provide a better lipid environment, i.e. how proteins differentially control their journey along the secretory pathway. Here we show by coarse-grained membrane simulations that proteins partition into the lipid phase with the smallest hydrophobic mismatch on heterogeneous membranes while they cluster and even segregate as homo-oligomers according to their hydrophobic mismatch on a homogeneous bilayer. We propose that protein sorting is facilitated by stabilizing coat proteins at clusters of mislocalized proteins that experience a hydrophobic mismatch.

Acylation is a frequent means to ensure membrane association of a variety of soluble proteins in living cells. However, many transmembrane proteins are palmitoylated, indicating that this posttranslational modification may also serve as a means to regulate protein trafficking. Based on coarse-grained membrane simulations, we find that protein acylation significantly alters the tilting of transmembrane proteins with respect to the bilayer normal. In addition, the proteins' partitioning behavior and cluster formation ability due to hydrophobic mismatching is strongly altered. Based on our results, we propose that acylation is a potent means to regulate the trafficking of transmembrane proteins along the early secretory pathway.

A multitude of transmembrane and secretory proteins enter the endoplasmic reticulum (ER) as unfolded polypeptide chains. In the ER, chaperones supervise the folding process and prevent unfolded proteins from entering anterograde transport intermediates, hence ensuring that only properly folded cargo is transported along the secretory pathway. It has been shown, however, that also unfolded proteins are very mobile in the ER thus questioning the model of a chaperone-mediated retention mechanism. To revisit this problem, we have used fluorescence correlation spectroscopy (FCS) on the temperature-sensitive tsO-45-G mutant of the vesicular stomatitis virus glycoprotein. Probing the short-range diffusional characteristics via FCS, we observed a strongly anomalous diffusion of tsO-45-G the in the ER in the unfolded state (at 39.5°C). The subdiffusion subsided when shifting to the permissive temperature (32°C) at which the protein is folded. Blocking the interaction of chaperones with the unfolded protein lead to a diffusion behavior similar to the folded protein. Based on our experimental results, we propose a model for the complex interplay between the chaperone machinery and unfolded membrane proteins.

Sorting of transmembrane proteins is a central task of eucaryotic cells, in particular in the secretory pathway.Due to a lack of an organizing mastermind the decision whether a membrane protein participates in secretory transport or not has to be made by a self-organization process on the molecular scale, e.g. via cluster formation. We show by means of coarse-grained membrane simulations that hydrophobic mismatching can drive cluster formation of transmembrane proteins [1]. Also, proteins with different degrees of hydrophobic mismatching can segregate and form homo-oligomers. In addition, we show that proteins partition into the lipid phase with the smallest hydrophobic mismatch if the membrane has a heterogeneous composition. Our data thus indicate that hydrophobic mismatching may help to organize trafficking along the secretory pathway in living cells.[1] U. Schmidt, G. Guigas & M. Weiss, Phys. Rev. Lett. 101, 128104 (2008).

Exit sites (ES) are specialized domains of the endoplasmic reticulum (ER) at which cargo proteins of the secretory pathway are packaged into COPII-coated vesicles. Although the essential COPII proteins (Sar1p, Sec23p-Sec24p, Sec13p-Sec31p) have been characterized in detail and their sequential binding kinetics at ER membranes have been quantified, the basic processes that govern the self-assembly and spatial organization of ERES have remained elusive. Here, we have formulated a generic computational model that describes the process of formation of ERES on a mesoscopic scale. The model predicts that ERES are arranged in a quasi-crystalline pattern, while their size strongly depends on the cargo-modulated kinetics of COPII turnover – that is, a lack of cargo leads to smaller and more mobile ERES. These predictions are in favorable agreement with experimental data obtained by fluorescence microscopy. The model further suggests that cooperative binding of COPII components, for example mediated by regulatory proteins, is a key factor for the experimentally observed organism-specific ERES pattern. Moreover, the anterograde secretory flux is predicted to grow when the average size of ERES is increased, whereas an increase in the number of (small) ERES only slightly alters the flux.

Macromolecular crowding provides the cytoplasm and the nucleoplasm with strongly viscoelastic properties and renders the diffusion of soluble proteins in both fluids anomalous. Here, we have determined the nanoscale viscoelasticity of the cytoplasm and the nucleoplasm in different mammalian cell lines. In contrast to the cell-specific response on the macroscale the nanoscale viscoelasticity (i.e. the behavior on length scales about 100-fold smaller than the cell size) only showed minor variations between different cell types. Similarly, the associated anomalous diffusion properties varied only slightly. Our results indicate a conserved state of macromolecular crowding in both compartments for a variety of mammalian cells with the cytoplasm being somewhat more crowded than the nucleus.

Underlying the nuclear envelope (NE) of most eukaryotic cells is the nuclear lamina, a meshwork consisting largely of coiled-coil nuclear intermediate filament proteins that play a critical role in nuclear organization and gene expression, and are vital for the structural stability of the NE/nucleus. By confocal microscopy and micromanipulation of the NE in living cells and isolated nuclei, we show that the NE undergoes deformations without large-scale rupture and maintains structural stability when exposed to mechanical stress. In conjunction with image analysis, we have developed theory for a two-dimensional elastic material to quantify NE elastic behaviour. We show that the NE is elastic and exhibits characteristics of a continuous two-dimensional solid, including connections between lamins and the embedded nuclear pore complexes. Correlating models of NE lateral organization to the experimental findings indicates a heterogeneous lateral distribution of NE components on a mesoscopic scale.

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D approximately equal to 15 microm(2) s(-1)), whereas coatomer diffuses 5-10 times more slowly than expected (D approximately equal to 1 microm(2) s(-1)). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.

To explain how resident proteins distribute in peak-like patterns at various positions in the secretory pathway, Glick and co-workers postulated that resident proteins comprise different populations (termed kin populations) and that these compete with each other for entering retrograde transport carriers [Glick et al. (1997) FEBS Lett. 414, 177–181]. Using modelling and computer simulation, they could demonstrate that differences in competitiveness sufficed to generate overlapping but distinct peak-like steady state distributions of the different kin populations across the Golgi stack. In this study, we have tested the robustness of the competition model and find that over-expression or changes in the number of kin populations affect their overall steady state distributions. To increase the robustness of the system, we have introduced a milieu-induced trigger for recycling. This allows for a decrease in the coupling between kin populations permitting both over-expression as well as changes in the number of kin populations. We have also extended the model to include a Golgi to endoplasmic reticulum (ER) recycling pathway and find that only a small amount of resident proteins may recycle at any time without upsetting their observed distributions in the Golgi stack. The biological relevance of a trigger-induced sorting mechanism and ER recycling is discussed.

Abstract 4079 The treatment and natural history of asymptomatic or smoldering myeloma has been an area of intense preclinical and clinical study. Historical attempts to treat these patients have not demonstrated significant benefit, likely as a consequence of limited therapeutic options as well lack of attention to the vast heterogeneity contained within the diagnosis of smoldering myeloma. More recent risk criteria from both US and Spanish investigators have identified a cohort of smoldering myeloma patients at high risk for progression to myeloma in a short time. Recently the PETHEMA group has conducted a randomized clinical trial testing lenalidomide and dexamethasone vs observation among high risk smoldering patients and has demonstrated a clear benefit in terms of progression free survival and hints towards improvement in overall survival favoring early intervention. To further evaluate the potential benefit of early intervention in a high risk smoldering cohort, the ECOG myeloma group designed a phase II trial testing the safety and efficacy of single agent lenalidomide with the intent of broadening to a randomized phase III trial if toxicity was acceptable. We report here on the safety portion of the phase II trial. The phase II group of patients received lenalidomide at a dose of 25 mg days 1–21 every 28 days to evaluate the early toxicity and tolerance of this dosing. The dose of lenalidomide could be adjusted based on toxicity using a defined dose-modification guideline in the protocol. The primary endpoint for the safety study was the rate of any treatment-related grade 4–5 non-hematologic toxicity plus grade 3 non-hematologic toxicity that affects vital organ function (such as cardiac, hepatic, or thromboembolic events) observed within 6 cycles of treatment. The goal was to enroll 34 patients; if 9 or more patients experience toxicity as defined, then the study would not continue. In terms of eligibility, patients were to be diagnosed with asymptomatic high-risk smoldering multiple myeloma (SMM) within the past 12 months, as confirmed by both of the following: bone marrow plasmacytosis with >= 10% plasma cells or sheets of plasma cells and an abnormal serum free light chain ratio (8.0) by serum FLC assay. Further, patients must have measurable levels of monoclonal protein (M-protein): >=1g/dL on serum protein electrophoresis or >=200 mg of monoclonal protein on a 24 hour urine protein electrophoresis. Patients must have no lytic lesions on skeletal surveys and no hypercalcemia. Among the 36 patients enrolled in the phase II study, 56% were female, and 44% were 65 years and older. Treatment and toxicity data at a minimum through cycle 6 is complete as of August 2, 2102. The last patient enrolled completed cycle 6 treatment on June 7, 2012. The median treatment duration of the entire cohort is 9 cycles (range 1–18 cycles), with 86% of patients completing at least 6 cycles of treatment. Ten patients are off treatment for the following reasons: disease progression (n=1), AE/complication (n=5), death (n=1) and patient withdrawal/refusal (n=3); 5 of the 10 patients ended treatment before completing 6 cycles. Of 36 patients assessed for toxicity, 8 patients [22.2%, 90% CI: (11.6%-36.5%)] experienced worst grade treatment-related non-hematologic toxicity of grade 3 or higher. Separately, 6 patients experienced unrelated non-hematologic toxicity of grade 3 or higher. Three patients [8.3%, 90% CI: (2.3%-20.2%)] experienced a serious adverse event as defined for the purposes of the phase II toxicity analysis (treatment-related grade 4–5 non-hematologic toxicity or grade 3 non-hematologic toxicity that affects vital organ function (such as cardiac, hepatic, or thromboembolic events). Based upon this analysis of the study, the phase II portion of the trial met the prespecified safety benchmark established to allow for phase III expansion, and accrual to the phase III portion of this study will begin. Efficacy data will be presented at the time of presentation. Disclosures: Lonial: Novartis: Consultancy; Millennium: Consultancy; Onyx: Consultancy; BMS: Consultancy; Celgene Corp: Consultancy; Merck: Consultancy. Off Label Use: Lenalidomide is not approved for treatment of smoldering MM. Fonseca:Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy; Binding site: Consultancy; Millenium: Consultancy; AMGEN: Consultancy; Celgene : Research Funding; Onyx: Research Funding; prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties. Dhodapkar:Celgene: Research Funding; KHK: Research Funding.

Abstract 3165 Background: Accrual to large oncology group studies in the United States is often slower than planned, especially in less common malignancies such as Multiple Myeloma (MM). To improve the treatment of patients with MM, it is essential that we address the barriers to accrual (BtA) to national clinical trials. The NCI Myeloma Steering Committee (MYSC) formed the Accrual Working Group (AWG), charged to analyze this problem. Methods: The members of the MYSC have developed a list of 10 potential BtA to NCI MM-CT under the leadership of the AWG at meetings conducted in person and by monthly telephone conference. This list was distributed via Survey Monkey among investigators and research staff of the SWOG, ECOG/ACRIN and Alliance research bases for the purpose of ranking the perceived importance of the 10 selected BtA. Responses based on a 0–5 Likert Scale were analyzed as continuous and categorical. For the latter, responses were grouped: Impedes Accrual – No (0,1), Yes (2–5); Significantly Impedes Accrual – No (0–3), Yes (4,5). Results: 246 survey responses were received. 50 participants who indicated that they had never attempted to or actually never enrolled a patient into a MM-CT were excluded. Of 196 responders, 61% had enrolled at least 5 patients, 43% practiced in academic centers (A) and 57% in community centers (C). 26% identified themselves as research staff, 74% as primarily physician investigators including Principal Investigators (12%). 78% have current access to NCI MM-CT. 46% identified their research base affiliation with SWOG, 20% with ECOG/ACRIN, 17% with Alliance, 17% as CTSU, NRG and other. The majority of survey responders confirmed agreement in the perception that all 10 identified BtA impede accrual, ranging from 60–84% depending on the specific barrier identified, while significant impediment to accrual ascribed to these BtA ranged from 22–47%. The perceived barriers most often cited as impeding accrual were (1) spectrum of available treatment options, (2) level of reimbursement for clinical trial related expenses and (3) patient‘s need for prompt treatment intervention (84%, 80%, 76% respectively). The first two barriers along with the requirement that patients receive their treatment exclusively at the NCI designated treatment site were rated most often as significantly impeding accrual (37%, 47%, 34% respectively). The first two were also identified most often as perceived BtA independent of practice setting (A, C), type of research engagement (physician investigator, research staff) and level of experience with MM-CT. Interestingly, the perception that patients must receive their treatment at the NCI designated treatment site and competing industry sponsored clinical trials were substantially more often cited as impeding accrual in academic centers compared to community centers and by physician investigators compared to research staff. The complexity of required diagnostic work up and the complexity of the treatment protocol and follow up testing were substantially more often cited as BtA in the community setting and by research staff compared to their peers in the academic setting and by physician investigators. The NCI MYSC AWG has proposed specific solutions to potentially lower perceived BtA. These include guidelines to allow protocol specific administration of commercially available agents by the patient‘s local oncologist, a simplified diagnostic work up and treatment plan and eligibility inclusion of patients who have received up to one cycle of specified urgent therapy prior to enrollment in MM-CT. Conclusions: We have queried experienced clinical investigators and research staff on 10 potential MM-CT specific BtA and found substantial consensus among responding participants in this survey. There are apparent differences in perceived BtA between the respondents from academic and community centers as well as between research staff and physician investigators. These data suggest that a structured approach to identification of BtA to clinical trials is feasible and creates opportunities to overcome them. This may have application to clinical trials across other diseases and may also lead to improvements in accrual. Disclosures: Barlogie: Celgene, IMF, MMRF, Millennium, Genzyme: Consultancy; Celgene, IMF, Millennium: Honoraria; Celgene, Novartis, NCI, Millennium, Onyx, Icon: Research Funding; GEP: Patents & Royalties.

Abstract 3647 Introduction: BR chemoimmunotherapy was shown to have an overall response rate (ORR) of 59%, a median progression-free survival (PFS) of 14.7 months, and an acceptable toxicity profile in R/R CLL (Fischer K, et al. J Clin Oncol 2011). Given the single-agent activity of lenalidomide in R/R CLL/SLL, we hypothesized that maintenance lenalidomide after BR induction could improve PFS. Methods: Thirty-four patients requiring therapy for R/R CLL/SLL were treated with bendamustine 90 mg/m2 IV on days 1 & 2 and rituximab 375 mg/m2 IV on day 1 every 28 days for a maximum of 6 cycles. Growth factor support was permitted. Patients achieving at least a minor response (objective improvement even if not meeting criteria for partial response) were eligible to proceed with 12 cycles of maintenance therapy with lenalidomide 5–10 mg/day orally given continuously in each 28-day cycle. Patients were eligible if they had histologically proven CLL/SLL and had received >1 but ≤5 prior cytotoxic chemotherapy regimens (retreatment with an identical regimen was not counted as a separate treatment). The primary endpoint was PFS. Results: Baseline characteristics include median age 67 (range 38–86), 25 men/9 women, 26 CLL/8 SLL, and median of 2 prior therapies (range 1–4). Cytogenetic profiling by FISH analysis was available in 22 patients (65%), with 11/22 demonstrating presence of 17p and/or 11q deletions. Twenty-five patients (74%) completed 6 cycles of induction BR. Two patients died from toxicities of pneumonia and heart failure during cycle 1; 7 patients received Conclusions: In our multicenter trial for patients with R/R CLL/SLL, the BR induction produced an ORR that is comparable to historical observations (65% vs 59%). However, the median PFS is longer (24.3 vs 14.7 months), suggesting maintenance lenalidomide may be contributing to an improved response duration. Based upon these promising results, we have initiated a successor study in which patients will receive lenalidomide plus rituximab maintenance after a BR induction. Disclosures: Chang: Celgene: Research Funding; Genentech: Research Funding. Off Label Use: Lenalidomide as maintenance therapy for CLL after induction rituximab + bendamustine chemoimmunotherapy. Fenske:Spectrum Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Kahl:Millennium: Consultancy, Research Funding; Roche: Consultancy; Genentech: Consultancy, Research Funding.