Complex Machinery Research Articles

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a ‘β-signal’ imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the β-signal, arranging several β-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'β-signal' imprinted in the final β-strand of the OMP engages the β-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the β-signal are repeated in other, internal β-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the β-signal, arranging several β-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

Somatostatin receptors and the associated intracellular machinery: the two sides of the coin.

Somatostatin receptors (SSTs) are widely expressed in pituitary tumors and neuroendocrine neoplasms (NENs) of different origins, i.e. the gastrointestinal tract and the thorax (lungs and thymus), thus representing a well-established target for medical treatment with SST ligands (SRLs). However, the response to SRLs is highly heterogeneous between tumors. Two main factors can contribute to this variability: (i) the differential SST expression among tumor types and (ii) the differential expression/modulation of the SST-related intracellular machinery. In this literature review, we provide an overview of available data on the variable expression of SSTs in pituitary tumors and NENs, together with the resulting clinical implications. Moreover, we aim to describe the complex intracellular machinery involved in SST signaling and trafficking. Particularly, we will focus on β-arrestins and describe their role in receptor internalization and recycling, as well as the various functions of these scaffold molecules in tumor pathogenesis and progression. This review highlights the interplay between membrane receptors and intracellular machinery, together with its role in determining the clinical behavior of the tumor and the response to treatment in patients with pituitary tumors or NENs.

Parallel structure of crayfish optimization with arithmetic optimization for classifying the friction behaviour of Ti-6Al-4V alloy for complex machinery applications

Intelligent techniques play a vital role in predicting the friction force during the wear of Ti-6Al-4V alloy under different lubricating conditions. The effective assessment of friction forces and lubricating conditions allows for the replacement of the material before catastrophic failure. However, it remains challenging to utilise friction forces under different lubrication conditions to predict the wear through intelligent techniques. In this work, an advanced technique based on artificial intelligence has been proposed to address this issue. Intially parallel structure of crayfish optimization and arithmetic optimization algorithm (PSCOAAOA) is developed to duly address the issues of slow convergence, stucking in local optima and quality of the solution. The PSCOAAOA is further implemented for finding the optimal parameters (regularization parameter and kernel function) of the Support Vector Machine (SVM). The quantitative and qualitative analysis of PSCOAAOA is carried out on CEC2014 benchmark functions to validate its efficacy and robustness. The friction force generated during wear testing under different lubricating conditions is bifurcated into training and test data. Out of which, training data trains the SVM at an optimal combination of parameters. The overall accuracy of the built SVM model is found to be 95.85% with a computation time of 26.85 s.

Dissecting the Organization of a β-Barrel Assembly Machinery (BAM) Complex by Neutron Reflectometry.

The β-barrel assembly machinery (BAM) complex in Gram-negative bacteria facilitates the assembly of β-barrel proteins into the outer membrane. Understanding the protein-protein interactions within this complex is essential for unravelling its functional mechanisms. Here, we present the use of neutron reflectometry for investigating the organization of β-barrel membrane protein complexes in the membrane environment. The spatial organization, protein positioning, protein-lipid interactions, and conformational changes within the complex can be elucidated by this method.

Design and test of a Human-Machine Interface for assembly lines in a learning factory

Human-Machine Interfaces are pivotal in modern manufacturing, serving as the critical link between human operators and complex machinery. This paper explores the evolution of HMIs, emphasizing their importance in enhancing efficiency, safety, and productivity within manufacturing environments. While HMIs offer real-time monitoring and control functionalities, their effectiveness hinges on human-centric design principles. Incorporating insights from human-centred design, this research presents a case study conducted at the SLIM laboratory of the University of Bergamo. The study encompasses theoretical exploration, HMI re-design, and user evaluation within an automatic assembly line setting. The research identifies key factors influencing HMI usability through critical analysis and user feedback and offers valuable insights for optimizing HMIs and empowering operators in the digital manufacturing landscape.

ОБЗОР СОВРЕМЕННЫХ ДЕМПФИРУЮЩИХ СПЛАВОВ В КОНТЕКСТЕ ПЕРСПЕКТИВ АГРОПРОМЫШЛЕННОГО ПРИМЕНЕНИЯ

The article provides an information and analytical review of progressive scientific and technical solutions in the field of industrial use of modern damping alloys from the point of view of increasing the effectiveness of vibration control and industrial noise in the operation of machinery and equipment of the agro-industrial complex. In accordance with the generalized classification system for high-damping alloys, a comprehensive characteristic of alloys with an improved ability to dissipate (absorb) the energy of vibrations that occur in dynamically loaded parts (nodes) of agricultural machinery is presented. The main groups of damping alloys for effective vibration protection of elements of agroengineering systems have been identified by systematizing scientific and practical information about the key features of the most common alloys in science and industry with increased damping (dissipative) properties. It has been established that such iron-carbon alloys as alloyed steels and gray cast irons are among the most promising vibration- and noise-absorbing metal materials for the manufacture of agrotechnical facilities operating in the mode of cyclic and shock effects. The indicated iron-carbon alloys have demonstrated an increased ability to dissipate the energy of sound vibrations over a wide temperature range with an acceptable set of physico-mechanical, technological and operational parameters. It was found out that the mass use of non-ferrous metal alloys in the agro-industrial complex as vibration- and noise-absorbing metal materials is hindered by their high cost, as well as a rather limited combination of physico-mechanical (functional) properties, especially in the range of elevated temperatures. For this reason, it is recommended to pay attention to the study, development and modernization of special multicomponent damping coatings that can be applied to the main material of agrotechnical facilities using various technologies.

РОМЕНСЬКЕ ТОВАРИСТВО СІЛЬСЬКИХ ГОСПОДАРІВ, ЙОГО ФІЛІЇ ТА ДОСЛІДНІ ПОЛЯ (1896–1914)

The place and role of the Romenske Rural Cooperative (hereinafter referred to as the Cooperative) and its branches in the reconstruction of the agricultural sector in Posullya are being explored. The Cooperative, established at the end of the 19th century, was one of the first in the region and became a unique phenomenon in agricultural research. From the early months of its activity, the Cooperative contributed to innovative improvements and the dissemination of agricultural technology, implementing progressive forms of management. Its established branches and research fields served as a link between science and practice. The Cooperative comprehensively promoted the development of agriculture in the region. It conducted significant educational work, addressed issues of credit acquisition for peasants, established a center for agricultural surplus, which not only sold products but also provided services for renting complex equipment and machinery. It actively organized agricultural exhibitions and fairs, encouraged farmers with awards, disseminated scientific literature, created a network of meteorological stations, mobile libraries, advisory-veterinary points, and more. The society, founded with the participation of local civic initiative, and its activities can serve as an example of reforming agro-industrial production in the context of the specific Posullya region. The Cooperative made a significant contribution to the development of scientific research, becoming an exemplar in implementing the gained experience into agricultural production.

In Vitro Reconstruction of Bacterial β-Barrel Membrane Protein Assembly Using E. coli Microsomal (Mid-Density) Membrane.

The in vitro reconstruction assay enables us to evaluate in detail the insertion and proper protein folding (together termed assembly) of β-barrel membrane proteins. Here, we introduce an in vitro reconstitution experiments using isolated membrane fractions from Escherichia coli(E. coli). Membrane fractions isolated from E. coli cells and disrupted by sonication, which we have termed E. coli microsomal (mid-density) membrane (EMM), are ideal for biochemical experiments, as they can be harvested by high-speed centrifugation and do not require ultra-centrifugation. EMM pretreated with detergent can assemble externally supplemented β-barrel membrane proteins via intact β-barrel assembly machinery (BAM) complex retained in EMM. This method not only allows assembly analysis with inexpensive equipment but it also can be applied to drug screening using assembly as an indicator with high reproducibility. In this chapter, we introduce our method of evaluating assembled β-barrel membrane proteins by demonstrating four representative β-barrel membrane proteins: E. coli major porins OmpA and OmpF; enterohemorrhagic E. coli (EHEC) autotransporter EspP, and Haemophilus influenzae (H. influenzae) adhesin Hia.

Role of heparin‐binding mediators in the resistance to anti‐VEGF therapies in diabetic retinopathy

Aims/Purpose: Anti‐vascular endothelial growth factor (VEGF) drugs represent the standard of care for diabetic retinopathy (DR); however, lack of response occurs in approximately 50% of patients. Aim of our work is the identification of the mediators responsible for anti‐VEGF drug resistance.Methods: Vitreous samples were collected from anti‐VEGF non‐responder (non‐RES) patients; non‐RES heat‐inactivated (HI) vitreous were used as control. To develop a new orthotopic model of DR, non‐RES or HI vitreous were injected intravitreally in mice and after 48 h retinas were explanted and analysed. The composition of non‐RES vitreous was assessed by an angio‐inflammatory protein array. The effect of the inhibition of the bioactive heparin‐binding mediators (HBMs) present in non‐RES vitreous was evaluated in the endothelial spheroid sprouting assay.Results: Mouse orthotopic intravitreal injection of non‐RES vitreous upregulated the expression of pro‐angiogenic/pro‐inflammatory mediators; moreover, it induced alterations of retinal vessels, accumulation of inflammatory infiltrate, and activation of astrocyte gliosis. Notably, HI vitreous was ineffective, pointing to a proteinaceous nature of the non‐RES vitreal mediators acting on mouse retinae. In this context, the blockade of vitreal HBMs mediated by the biotechnological heparin‐like molecule K5‐N, OS (H) or by the pentapeptide Boc2, completely abrogated the activity of non‐RES samples. It is worth noticing that the potency of anti‐HBP compounds was significantly higher than the anti‐VEGF ranibizumab, which was partially effective only at the clinically relevant and saturating concentration of 10 μM, with no further improvement at a double dose.Conclusions: Our data show that VEGF is only part of a complex machinery regulating angiogenesis, inflammation and oedema formation during DR and that drug resistance is due to the activation of compensatory mechanisms mediated by HBMs besides VEGF.

Extracellular vesicles and cancer stem cells: a deadly duo in tumor progression.

The global incidence of cancer is increasing, with estimates suggesting that there will be 26 million new cases and 17 million deaths per year by 2030. Cancer stem cells (CSCs) and extracellular vesicles (EVs) are key to the resistance and advancement of cancer. They play a crucial role in tumor dynamics and resistance to therapy. CSCs, initially discovered in acute myeloid leukemia, are well-known for their involvement in tumor initiation, progression, and relapse, mostly because of their distinct characteristics, such as resistance to drugs and the ability to self-renew. EVs, which include exosomes, microvesicles, and apoptotic bodies, play a vital role in facilitating communication between cells within the tumor microenvironment (TME). They have a significant impact on cellular behaviors and contribute to genetic and epigenetic changes. This paper analyzes the mutually beneficial association between CSCs and EVs, emphasizing their role in promoting tumor spread and developing resistance mechanisms. This review aims to investigate the interaction between these entities in order to discover new approaches for attacking the complex machinery of cancer cells. It highlights the significance of CSCs and EVs as crucial targets in the advancement of novel cancer treatments, which helps stimulate additional research, promote progress in ideas for cancer treatment, and provide renewed optimism in the effort to reduce the burden of cancer.

In Vitro Kinase Assay with Recombinant SnRK2s: An Example for Assaying Stress-Responsive Kinases in Plants.

Protein phosphorylation is one of the most important posttranslational modifications in cell signaling pathways. Kinases and phosphatases play essential roles in transferring information between sensors and effectors under stress conditions. Several methods have been developed to analyze the phosphorylation mechanisms. Each method has advantages and disadvantages. In vitro kinase assay using recombinant proteins is a method to analyze kinase activities under simplified conditions. It is a good strategy to understand each mechanism one by one, although it is not always suitable to estimate the feature of complex machinery in vivo. In this chapter, the purification of recombinant proteins produced in Escherichia coli followed by assaying a kinase activity using radioactivity is described.

A framework to model contractors’ hazard and risk exposure at process plants using unsupervised text mining

Increasing outsourcing of operations in process industries is leading to contractors facing higher exposure to complex processes and machinery. This necessitates development of safety management systems by performing fault detection and diagnosis, accident/failure modelling and risk assessment of contractors’ operations and associated hazards. Unfortunately, limited work has been done in this area. Moreover, in broad process safety domain, using text mining to perform accident modelling and risk assessment has not been fully exploited. We address these research gaps by proposing an unsupervised text mining framework to generate accident/failure models like chain of events and fault trees from investigation reports of contractor incidents. A case study is used to demonstrate the framework where incident investigation reports of contract personnel working at a steel plant in India are analysed using a combination of topic modelling and association rule mining. Twenty sequences of basic events are identified as major chains of events. These chains of events are then combined using logic rules to generate fault trees depicting all possible propagation paths of contractor safety failures leading to top events. Our framework enables generation of chain of events and fault trees from unstructured incident reports with minimal human supervision with accuracy of 81%.

High Densification Level and Hardness Values of Additively Manufactured 316L Stainless Steel Fabricated by Fused Filament Fabrication

Laser powder bed fusion (L-PBF) has emerged as the most widely used additive manufacturing (AM) process, also known as 3D printing, to fabricate 316L stainless steel (316L SS) components for various applications. However, the initial setup, operation, and maintenance costs are too expensive due to the complex machinery, high energy-consuming laser beam, and proprietary software required. Therefore, in this paper, fused fiament fabrication (FFF) is proposed as a low-cost AM approach to fabricate 316L SS specimens via a 3-step printing-debinding-and sintering process. The specimens are initially printed on a desktop FFF AM 3D printer by varying nozzle temperatures from 195 – 220°C, followed by debinding up to 427°C for 4 hours, and finally sintering at 1260°C for 4 hours. The results show that nozzle temperature 200°C yielded the highest densification level of 97.6% and highest average hardness value of 292 HV, indicating that 3D printing parameters, particularly nozzle temperature plays an important role in influencing the properties of the sintered specimens. Overall, the results from this study prove that FFF is a viable and cost-effective AM process that has the potential to produce 316L SS parts that meet industrial requirements.

Counting and Localization of Multiple Unknown Harmonic Sound Sources in Entire 3D Space Using Just Five Microphones

Counting and localization of multiple sound sources in an unknown environment has many applications, namely fault diagnosis of complex machineries, hearing aids' development, and localization of enemy vehicles in defense. However, multiple sound source localization is extremely challenging because it requires numerous microphones, which increases the cost, computational time, and installation difficulties. Here, we present a new method to count the number of stationary harmonic sound sources and localize them in a free-field environment using a novel oblique square pyramid shaped array of only 5 microphones. We find FFT peaks of microphone signals and compare the ratio of the magnitude of the most dominant peak with remaining peaks' one-by-one. If these ratios are below 25 dB difference, they are considered as sound source and counted. A new time delay and intensity ratio computation is applied to the segregated source signals to calculate exact coordinates of each source. Simulation studies of four concurrent sources show that our method predicts all sources' coordinates at every octant location with mean error of 3.4%, and less than 5% error beyond 20 dB Signal-to-Noise ratio. Mean computational time is 0.92 second. Therefore, our method is low-cost, quicker, more accurate and more reliable than existing techniques.

Stress-Induced AMPK Interactome Analysis Reveals Novel Protein-Protein Interactions Associated with Chromatin Remodeling and Transcription Machinery in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death among children, adolescents and young adults. Survival rates for relapsed/refractory ALL remain dismal. We have previously reported that ALL cells are vulnerable to energy/metabolic stress conditions following AMP-activated protein kinase (AMPK) activation, leading to cell death. AMPK is the master metabolic regulator, and our lab and others have reported it interacts with chromatin-associated proteins to epigenetically regulate gene expression in response to energy/metabolic stress. To identify genome-wide genes regulated by direct association of AMPK to chromatin in response to energy/metabolic stress, we previously used ChIP-seq and RNA-seq in Bp-ALL (KASUMI-2) and T-ALL (KE-37) cells treated with or without glucose or AICAR, a well-known AMPK activator, and found that (i) AMPK interacts with components of the transcription machinery including the TATA-Box Binding Protein Associated Factor (TAF) and RNA polymerase II, (ii) AMPKα2 was enriched for genes involved in histone H3-K4 methylation, protein localization, Notch signaling, and mRNA destabilization, (iii) AMPKa2 was recruited to promoter regions of the epigenetic regulators KMT2A and SETD1A, and (iv) the mRNA expression of KMT2A and SETD1A genes was upregulated in KASUMI-2 cells treated with AICAR. To further investigate AMPK's involvement in epigenetic mechanisms in response to energy/metabolic stress, we performed a comprehensive interactome analysis using TurboID-based proximity labeling proteomics. AMPK is a highly conserved heterotrimeric kinase complex composed of catalytic α-subunit (α1 or α2) and regulatory β (β1 or β2) and γ (γ1, γ2, or γ3) subunits. We generated HEK293T cells stably expressing TurboID fused to the AMPKα1, AMPKα2, AMPKβ1 or AMPKγ1 (which are the most abundant AMPK subunits expressed in ALL cells), and these stable cell lines were treated with the potent allosteric AMPK activator PF-06409577 for 1 hr, to mimic the cell's rapid response to energy/metabolic stress. Following characterization of these stable cell lines and small-scale optimization experiments, treated samples and controls were submitted to mass spectrometry analysis. Proteomics data analysis identified novel AMPK's protein-protein interactions (PPI) which were altered (enhanced or reduced) following PF-06409577 treatment. Among PPI altered by PF-06409577 treatment, 148 were enhanced and 144 reduced for AMPKα1, 169 enhanced and 172 reduced for AMPKα2, 250 enhanced and 188 reduced for AMPKβ1 and 289 enhanced and 153 reduced for AMPKγ1. For proteomics data visualization we used Cytoscape software. The GO analysis of proteins interacting with AMPKα1β1γ1 and/or AMPKα2β1γ1 heterotrimeric complexes in cells treated with PF-06409577 showed that energy/metabolic stress enhances their interaction with proteins involved in gene expression, cellular metabolic processes, chromatin organization, DNA repair, histone modifications, etc.. Using the STRING database, we performed MCL clustering analysis and uncovered a major cluster of known epigenetic regulators (e.g., histone modifiers such as KDM6A, HDAC2, SETD2, KMT2A and SETD1A etc.) and components of the transcription machinery (such as NELFE, POLR2A etc.) that interacted with AMPK under stress-induced conditions. These data support our previous findings uncovered by ChIP-seq and RNA-seq analysis. Consequently, our findings further validate an epigenetic role for AMPK via interactions with putative members of chromatin-associated and transcription machinery complexes and chromatin modifying enzymes that allow ALL cells to respond to conditions of energy/metabolic stress. To our knowledge, this is the first report describing the AMPK interactome in pediatric ALL. Further elucidation of the uncovered protein-protein interactions may lead to potential epigenetic-based targeted therapies to overcome therapeutic resistance in relapse/refractory ALL and other hematological malignancies.

Complement System and the Kidney: Its Role in Renal Diseases, Kidney Transplantation and Renal Cell Carcinoma.

The crosstalk among the complement system, immune cells, and mediators of inflammation provides an efficient mechanism to protect the organism against infections and support the repair of damaged tissues. Alterations in this complex machinery play a role in the pathogenesis of different diseases. Core complement proteins C3 and C5, their activation fragments, their receptors, and their regulators have been shown to be active intracellularly as the complosome. The kidney is particularly vulnerable to complement-induced damage, and emerging findings have revealed the role of complement system dysregulation in a wide range of kidney disorders, including glomerulopathies and ischemia-reperfusion injury during kidney transplantation. Different studies have shown that activation of the complement system is an important component of tumorigenesis and its elements have been proved to be present in the TME of various human malignancies. The role of the complement system in renal cell carcinoma (RCC) has been recently explored. Clear cell and papillary RCC upregulate most of the complement genes relative to normal kidney tissue. The aim of this narrative review is to provide novel insights into the role of complement in kidney disorders.

Research into a Marine Helicopter Traction System and Its Dynamic Energy Consumption Characteristics

As countries attach great importance to the ocean-going navigation capability of ships, the energy consumption of shipborne equipment has attracted much attention. Although energy consumption analysis is a guiding method to improve energy efficiency, it often ignores the dynamic characteristics of the system. However, the traditional dynamic analysis method hardly considers the energy consumption characteristics of the system. In this paper, a new type of electric-driven helicopter traction system is designed based on the ASIST system. Combined with power bond graph theory, a system dynamic modeling method that considers both dynamic and energy consumption characteristics is proposed, and simulation analysis is carried out. The results indicate that the designed traction system in this study displays high responsiveness, robust, steady-state characteristics, and superior energy efficiency. When it engages with helicopter-borne aircraft, it swiftly transitions to a stable state within 0.2 s while preserving an efficient speed tracking effect under substantial load force, and no significant fluctuations are detected in the motor rotation rate or the helicopter movement velocity. Moreover, it presents a high energy utilization rate, achieving an impressive energy utilization rate of 84% per single working cycle. Simultaneously, the proposed modeling methodology is validated as sound and effective, particularly apt for the dynamic and power consumption analysis of marine complex machinery systems, guiding the high-efficiency design of the transmission system.

Enteroendocrine cell regulation of the gut-brain axis.

Enteroendocrine cells (EECs) are an essential interface between the gut and brain that communicate signals about nutrients, pain, and even information from our microbiome. EECs are hormone-producing cells expressed throughout the gastrointestinal epithelium and have been leveraged by pharmaceuticals like semaglutide (Ozempic, Wegovy), terzepatide (Mounjaro), and retatrutide (Phase 2) for diabetes and weight control, and linaclotide (Linzess) to treat irritable bowel syndrome (IBS) and visceral pain. This review focuses on role of intestinal EECs to communicate signals from the gut lumen to the brain. Canonically, EECs communicate information about the intestinal environment through a variety of hormones, dividing EECs into separate classes based on the hormone each cell type secretes. Recent studies have revealed more diverse hormone profiles and communication modalities for EECs including direct synaptic communication with peripheral neurons. EECs known as neuropod cells rapidly relay signals from gut to brain via a direct communication with vagal and primary sensory neurons. Further, this review discusses the complex information processing machinery within EECs, including receptors that transduce intraluminal signals and the ion channel complement that govern initiation and propagation of these signals. Deeper understanding of EEC physiology is necessary to safely treat devastating and pervasive conditions like irritable bowel syndrome and obesity.

Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM

The β-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by “slow” and “fast” diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.