Recycling Of Membrane Proteins Research Articles

Required minimal protein domain of flower for synaptobrevin2 endocytosis in cytotoxic T cells

Flower, a highly conserved protein, crucial for endocytosis and cellular fitness, has been implicated in cytotoxic T lymphocyte (CTL) killing efficiency through its role in cytotoxic granule (CG) endocytosis at the immune synapse (IS). This study explores the molecular cues that govern Flower-mediated CG endocytosis by analyzing uptake of Synaptobrevin2, a protein specific to CG in mouse CTL. Using immunogold electron microscopy and total internal fluorescence microscopy, we found that Flower translocates in a stimulus-dependent manner from small vesicles to the IS, thereby ensuring specificity in CG membrane protein recycling. Using confocal live-cell imaging, we assessed the ability of a range of naturally occurring mouse, human and Drosophila isoforms to rescue defective endocytosis in Flower KO CTLs. This analysis demonstrated that the N-terminal portion of the protein, encompassing amino acids 1–106 in mice, is the minimal domain necessary for Synaptobrevin2 endocytosis. Additionally, we identified two pivotal sites through site-specific mutation: a putative AP2-binding site, and a tyrosine at position 104 in mouse Flower. These findings provide insights into Flower's specific functional domain essential for CG endocytosis, which is a key process in mediating T cell serial killing required for the effective fight against cancer.

Endo-IP and lyso-IP toolkit for endolysosomal profiling of human-induced neurons

Plasma membrane protein degradation and recycling are regulated by the endolysosomal system, wherein endocytic vesicles bud from the plasma membrane into the cytoplasm and mature into endosomes and then degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface and influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we develop a toolkit for capturing EEA1-positive endosomes (termed Endo-IP) and TMEM192-positive lysosomes (termed Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrate its utility by revealing the endolysosomal protein landscapes for stem cells and cortical-like iNeurons, and profiling endosomes in response to potassium-mediated neuronal depolarization. Through global profiling of endocytic cargo, we identify hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.

Plasma membrane protein degradation and recycling is regulated by the endolysosomal system, wherein endosomes bud from the plasma membrane into the cytosol and mature into degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface, influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we developed a toolkit for capturing EEA1-postive endosomes (Endo-IP) and TMEM192-positive lysosomes (Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrated its utility by revealing the endolysosomal protein landscapes for cortical-like iNeurons and stem cells. This allowed us to globally profile endocytic cargo, identifying hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

Effect of CFTR Modulators on Oxidative Stress and Autophagy in Non-CFTR-Expressing Cells.

The triple combination therapy for cystic fibrosis (CF), including elexacaftor, tezacaftor and ivacaftor (ETI or Trikafta), has been shown to improve lung function and reduce pulmonary exacerbations, thereby enhancing the quality of life for most CF patients. Recent findings suggest that both the individual components and ETI may have potential off-target effects, highlighting the need to understand how these modulators impact cellular physiology, particularly in cells that do not express CF transmembrane conductance regulator (CFTR). We used HEK293 cells, as a cell model not expressing the CFTR protein, to evaluate the effect of ETI and each of its components on autophagic machinery and on the Rab5/7 components of the Rab pathway. We firstly demonstrate that the single modulators Teza and Iva, and the combinations ET and ETI, increased ROS production in the absence of their target while decreasing it in cells expressing the CFTR ∆F508del. This increase in cellular stress was followed by an increase in the total level of polyubiquitinated proteins as well as the p62 level and LC3II/LC3I ratio. Furthermore, we found that ETI had the opposite effect on Rabs by increasing Rab5 levels while decreasing Rab7. Interestingly, these changes were abolished by the expression of mutated CFTR. Overall, our data suggest that in the absence of their target, both the individual modulators and ETI increased ROS production and halted both autophagic flux and plasma membrane protein recycling.

Real-time imaging of axonal membrane protein life cycles.

The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

Quantitative Measurement of Plasma Membrane Protein Internalisation and Recycling in Heterogenous Cellular Samples by Flow Cytometry.

Plasma membrane proteins mediate important aspects of physiology, including nutrient acquisition, cell-cell interactions, and monitoring homeostasis. The trafficking of these proteins, involving internalisation from and/or recycling back to the cell surface, is often critical to their functions. These processes can vary among different proteins and cell types and states and are still being elucidated. Current strategies to measure surface protein internalisation and recycling are typically microscopy or biochemical assays; these are accurate but generally limited to analysing a homogenous cell population and are often low throughput. Here, we present flow cytometry-based methods involving probe-conjugated antibodies that enable quantification of internalisation or recycling rates at the single-cell level in complex samples. To measure internalisation, we detail an assay where the protein of interest is labelled with a specific antibody conjugated to a fluorescent oligonucleotide-labelled probe. To measure recycling, a specific antibody conjugated to a cleavable biotin group is employed. These probes permit the differentiation of molecules that have been internalised or recycled from those that have not. When combined with cell-specific marker panels, these methods allow the quantitative study of plasma membrane protein trafficking dynamics in a heterogenous cell mixture at the single-cell level. Key features • These assays allow sensitive quantification of internalised or recycled surface molecules using oligonucleotide or cleavable biotin-conjugated probes, respectively, and detected by flow cytometry. • They can be adapted to any membrane protein that transits via the cell surface and for which a specific purified antibody is available. • The dynamics of a cell surface protein can be measured in heterogenous cell populations simultaneously, including various cellular activation states. • The internalisation assay builds upon the method developed by Liu et al. [1,2] and extends its application to heterogenous human peripheral blood mononuclear cells. • These assays have been extensively used on suspension cells but have not been tested on adherent cells.

TMED2 promotes glioma tumorigenesis by being involved in EGFR recycling transport

Aberrant epidermal growth factor receptor (EGFR) signaling is the core signaling commonly activated in glioma. The transmembrane emp24 protein transport domain protein 2 (TMED2) interacts with cargo proteins involved in protein sorting and transport between endoplasmic reticulum (ER) and Golgi apparatus. In this study, we found the correlation between TMED2 with glioma progression and EGFR signaling through database analysis. Moreover, we demonstrated that TMED2 is essential for glioma cell proliferation, migration, and invasion at the cellular levels, as well as tumor formation in mouse models, underscoring its significance in the pathobiology of gliomas. Mechanistically, TMED2 was found to enhance EGFR-AKT signaling by facilitating EGFR recycling, thereby providing the initial evidence of TMED2's involvement in the membrane protein recycling process. In summary, our findings shed light on the roles and underlying mechanisms of TMED2 in the regulation of glioma tumorigenesis and EGFR signaling, suggesting that targeting TMED2 could emerge as a promising therapeutic strategy for gliomas and other tumors associated with aberrant EGFR signaling.

Structural organization of the retriever-CCC endosomal recycling complex.

The recycling of membrane proteins from endosomes to the cell surface is vital for cell signaling and survival. Retriever, a trimeric complex of vacuolar protein-sorting-associated protein (VPS)35L, VPS26C and VPS29, together with the CCC complex comprising coiled-coil domain-containing (CCDC)22, CCDC93 and copper metabolism domain-containing (COMMD) proteins, plays a crucial role in this process. The precise mechanisms underlying retriever assembly and its interaction with CCC have remained elusive. Here, we present a high-resolution structure of retriever in humans determined using cryogenic electron microscopy. The structure reveals a unique assembly mechanism, distinguishing it from its remotely related paralog retromer. By combining AlphaFold predictions and biochemical, cellular and proteomic analyses, we further elucidate the structural organization of the entire retriever-CCC complex across evolution and uncover how cancer-associated mutations in humans disrupt complex formation and impair membrane protein homeostasis. These findings provide a fundamental framework for understanding the biological and pathological implications associated with retriever-CCC-mediated endosomal recycling.

P97/VCP Promotes the Recycling of Endocytic Cargo.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

Arabidopsis transmembrane proteins regulate protein turnover as signaling molecules during internalization by means of ubiquitination

<p>This study attempts to characterize the structure, function, and other features of Arabidopsis thaliana based on its transmembrane protein amino acid sequence and assess its potential as a drug target and ubiquitination site. The transmembrane protein belongs to the Arabidopsis plant, but little is known about the structure-function of the protein, among others. We predicted the structure and transmembrane domains of the transmembrane protein by different bioinformatic tools, and the prediction results showed that its three poorly conserved transmembrane domains could be used as potential drug targeting sites. Among them, lysine ubiquitination sites play an important role in the recycling of plant membrane proteins, and we found the existence of ubiquitination sites by predicting the ubiquitination sites of transmembrane proteins, and this could be a new research direction in the future.<strong></strong></p>

Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n= 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n= 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

Involvement of mTOR pathway in neurodegeneration in NSF-related developmental and epileptic encephalopathy.

Membrane fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. During neurotransmitter exocytosis, SNARE proteins on a synaptic vesicle and the target membrane form a complex, resulting in neurotransmitter release. N-ethylmaleimide-sensitive factor (NSF), a homohexameric ATPase, disassembles the complex, allowing individual SNARE proteins to be recycled. Recently, the association between pathogenic NSF variants and developmental and epileptic encephalopathy (DEE) was reported; however, the molecular pathomechanism of NSF-related DEE remains unclear. Here, three patients with de novo heterozygous NSF variants were presented, of which two were associated with DEE and one with a very mild phenotype. One of the DEE patients also had hypocalcemia from parathyroid hormone deficiency and neuromuscular junction impairment. Using PC12 cells, a neurosecretion model, we show that NSF with DEE-associated variants impaired the recycling of vesicular membrane proteins and vesicle enlargement in response to exocytotic stimulation. In addition, DEE-associated variants caused neurodegenerative change and defective autophagy through overactivation of the mammalian/mechanistic target of rapamycin (mTOR) pathway. Treatment with rapamycin, an mTOR inhibitor or overexpression of wild-type NSF ameliorated these phenotypes. Furthermore, neurons differentiated from patient-derived induced pluripotent stem cells showed neurite degeneration, which was also alleviated by rapamycin treatment or gene correction using genome editing. Protein structure analysis of NSF revealed that DEE-associated variants might disrupt the transmission of the conformational change of NSF monomers and consequently halt the rotation of ATP hydrolysis, indicating a dominant negative mechanism. In conclusion, this study elucidates the pathomechanism underlying NSF-related DEE and identifies a potential therapeutic approach.

CCDC22 and CCDC93, two potential retriever-interacting proteins, are required for root and root hair growth in Arabidopsis.

Endomembrane trafficking is essential for plant growth and often depends on a balance between secretory and endocytic pathways. VPS26C is a component of the retriever complex which has been shown to function in the recycling of integral plasma membrane proteins in human cell culture and is part of a core retriever complex in Arabidopsis that is required for root hair growth. In this work, we report a characterization of the Arabidopsis homologues of CCDC22 and CCDC93, two additional proteins required for retriever function in humans. Phylogenetic analysis indicates that CCDC22 (AT1G55830) and CCDC93 (AT4G32560) are single copy genes in plants that are present across the angiosperms, but like VPS26C, are absent from the grasses. Both CCDC22 and CCDC93 are required for root and root hair growth in Arabidopsis and localize primarily to the cytoplasm in root epidermal cells. Previous work has demonstrated a genetic interaction between VPS26C function and a VTI13-dependent trafficking pathway to the vacuole. To further test this model, we characterized the vti13 ccdc93 double mutant and show that like vps26c, ccdc93 is a suppressor of the vti13 root hair phenotype. Together this work identifies two new proteins essential for root and root hair growth in plants and demonstrate that the endosomal pathway(s) in which CCDC93 functions is genetically linked to a VTI13-dependent trafficking pathway to the vacuole.

Autophagosomal components recycling on autolysosomes.

Autophagy is a multistage, intracellular process. Here, we highlight a recently identified autophagosomal components recycling (ACR) stage and the recycler complex (SNX4-SNX5-SNX17), which mediates recycling of autophagosomal outer membrane proteins on the autolysosome surface immediately following autophagosome-lysosome fusion. This discovery opens numerous research directions into the postfusion fate of autophagosomes.

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by β and γ-Secretases, which produce amyloidogenic Aβ species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1.

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.

Tex264 Binding to SNX27 Regulates Itgα5 Receptor Membrane Recycling and Affects Cell Migration.

Tex264 is an endoplasmic reticulum (ER) membrane protein that was recently demonstrated to act as an ER-phagy receptor under starvation conditions to mediate endoplasmic reticulum autophagy. However, how Tex264 functions in the central nervous system (CNS) and tumors is unclear. Here, we identified 89 proteins from the rat brain that may specifically interact with Tex264 and confirmed the interaction between sorting nexin 27 (SNX27) and Tex264 by coimmunoprecipitation and immunofluorescence. Our results indicated that Tex264 may promote recycling of membrane proteins from endosomes to the cell plasma membrane by recruiting SNX27 retromer vesicles. siRNA-mediated knockdown of TEX264 in HeLa cells did not affect cell proliferation but did significantly inhibit cell migration through a mechanism that may involve a reduction in SNX27-mediated Itgα5 receptor membrane recycling. Results of this study helped identify potential binding Tex264 partners and provide insights into Tex264 functions in the CNS and in tumors.

Contribution of the Golgi and Endosomes to Proteostasis During Aging in the Nematode Caenorhabditis elegans

A gradual breakdown in the cellular networks that assure protein stability, termed proteostasis, contributes significantly to the accumulation of misfolded and aggregated proteins that severely compromises cellular function later in life. A progressive decline in the efficiency of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) causes cells to become particularly susceptible to disturbances affecting the biogenesis, folding, stability, and turnover of membrane proteins. While the dynamics of ER stress response mechanisms during aging have been well‐characterized, less is understood about how the function of other organelles important for membrane protein biogenesis and turnover might change over time. We hypothesized that age‐dependent changes in the function of the Golgi and endosomes could contribute to the collapse in proteostasis during adulthood. Using a reverse genetic approach in Caenorhabditis elegans, we probed the function of these two compartments by knocking down the expression of cogc‐2, a member of the Conserved Oligomeric Golgi (COG) complex and of rme‐8, encoding an endosomal protein important for the retrieval of Golgi residents and the recycling of plasma membrane proteins. A key component of the C. elegans innate immune response is the secretion of immune effector proteins into the intestinal lumen, which can itself trigger the ER stress response due to the increased flux through the secretory pathway. Therefore, as a proxy for the integrity of vesicular transport pathways we measured the survival of juvenile and adult animals infected with the bacterial pathogen Pseudomonas aeruginosa following RNAi treatments targeting cogc‐2 or rme‐8. While there was a negligible effect on late larval stage animals, reduced expression of cogc‐2 and rme‐8 enhanced the sensitivity of post‐reproductive adult animals to infection. This suggests that components of vesicular transport pathways downstream of the ER could also contribute to proteostasis in an age‐dependent manner through their roles in ensuring the proper localization of membrane proteins. Follow‐up studies will use in vivo fluorescent reporters of protein stability to determine whether interfering with protein transport through the Golgi and endosomes accelerates the decline in proteostasis in older animals. Our studies imply that organelles involved in late steps of membrane protein biogenesis and stability play increasingly important roles in maintaining the proteome during aging.

Impact of glycosylation on uptake of receptors by endocytosis

Clathrin-mediated endocytosis is a fundamental cellular uptake process that is essential for nutrient import, signaling, and recycling of membrane proteins. Many membrane proteins that are internalized by endocytosis are also glycosylated on their extracellular domains. Some of these glycosylated proteins are constituents of the glycocalyx, a cellular structure that provides a physical barrier to exclude pathogens from cells and maintains membrane hydration. Despite the essential role of membrane traffic in maintaining the glycocalyx, little is known about the impact of glycosylation on the uptake of membrane proteins.

Uptake of platelets by cancer cells and recycling of the platelet protein CD42a

BackgroundIt is well accepted that the bidirectional crosstalk between platelets and cancer cells promotes tumorigenesis and metastasis. In an early step, cancer cells trigger platelet granule and extracellular vesicle release that is needed to facilitate cancer cell survival in circulation. ObjectivesTo discover the early crosstalk of cancer cells and platelets. MethodsCancer cells were incubated with freshly isolated and stained human platelets. Confocal laser scanning microscopy and flow cytometry was used to visualize and to quantify platelet uptake and the membrane presence of CD42 on cancer cells. Dyngo4a was used to test if platelet uptake is a dynamin‐dependent process. ResultsWe found a dynamin‐dependent uptake of platelets by cancer cells. This is followed by the recycling of the platelet‐specific protein CD42a and its incorporation into cancer cells’ plasma membrane, which is not a result of platelet RNA transfer by platelet‐derived microparticles and exosomes. Time course of platelet uptake follows a sigmoid function revealing that 50% of the cancer cells are positive for platelets after approximately 38 min. Platelet uptake was observed for the tested cancerous cells (A549, MCF‐7, and MV3) but not for the non‐cancerous cell line 16HBE14o−. ConclusionsOur results demonstrate that cancer cells hijack platelets by phagocytosis and recycling of platelet membrane proteins. The uptake of platelets has additional advantages for cancer cells: access to the entire and undiluted platelet proteome, transcriptome, and secretome. These novel findings will allow further mechanistic elucidation and thus help us gain deeper insights into platelet‐assisted hematogenous metastasis.