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Publications and source records attributed to Phan, A..
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Cell states are increasingly conceptualized as attractors of high-dimensional dynamical systems, yet quantitative approaches for integrating phenotypic information into this framework remain limited. Here, we take an image-based approach that combines unsupervised machine learning (ML) with timelapse imaging to extract and characterize the temporal dynamics of morphological features. Using a cell line with endogenously tagged VE-cadherin, we acquired brightfield and fluorescence timelapse images of human induced pluripotent stem cell-derived endothelial cell (hiPSC-EC) monolayers, which adopt distinct phenotypes at two different magnitudes of shear stress in terms of their morphology, behavior, and VE-cadherin organization. To quantify these phenotypic cell states without segmentation, we trained a diffusion autoencoder to predict VE-cadherin signal from brightfield images. We identified interpretable ML-based features representing cell orientation, elongation, and local density. Treating these variables as dimensions of a morphological state space, we estimated a data-driven vector field and found that the two observed phenotypic cell states correspond to stable fixed points of the inferred dynamical system. Mapping measured cell migration coherence onto this space further distinguished the states. Imaging cells across intermediate shear stresses revealed a regime of bistability in which both states coexist, indicating that the shear-stress-dependent transition between endothelial cell states occurs as a bifurcation of the inferred dynamical system. Finally, we applied this method to study an N-terminal truncation of VE-cadherin, finding that mutated cells preserve alignment and coherent migration, but exhibit altered morphology and increased migration speed. This work demonstrates the applicability of a dynamical systems approach to quantitatively characterize morphological aspects of cell state from interpretable ML-based features.
MotivationHigh-throughput experiments generate genome-wide measurements for thousands of genes, which are often tested marginally. Biological processes are driven by coordinated groups of genes rather than individual genes, making gene set enrichment analysis an essential post hoc interpretation tool. Traditional approaches such as Over-Representation Analysis and Gene Set Enrichment Analysis test gene sets independently, which ignores the hierarchical and overlapping structure of gene set collections such as the Gene Ontology, and often leads to redundant enrichment results. Set-based approaches such as MGSA address this issue by modeling multiple gene sets simultaneously, but they rely on binary gene activation states derived from arbitrary thresholds on gene-level statistics. ResultsWe introduce Concise List Enrichment Analysis Reducing Redundancy (CLEAR), a Bayesian gene set enrichment framework that jointly models gene sets while incorporating continuous gene-level statistics such as test statistics or p-values. CLEAR extends model-based gene set analysis by replacing threshold-based gene activation with a probabilistic model for continuous gene-level statistics. This approach preserves the redundancy-reduction advantages of set-based enrichment methods while avoiding the information loss introduced by binarization. Using both simulated datasets and human gene expression data, we show that CLEAR improves sensitivity compared with existing enrichment approaches while producing a more concise and interpretable set of enriched gene sets. Availability and implementationThe source code, data, and a brief tutorial are freely available at https://github.com/jiatuya/CLEAR
Somatic recombination can have profound consequences including clonal evolution. In plants, however, the contribution of homologous recombination (HR) to genomic instability remains unclear. The semidominant sulfur mutant of tobacco, named for its yellow foliage resulting from a chlorophyll defect, spontaneously develops green and white twin spots that have been attributed to mitotic crossovers. To test this hypothesis, we sequenced DNA from twin spots and surrounding tissues in su/+ F1 hybrids, expecting somatic crossovers to result in reciprocal chromosome arm exchange in the paired sectors. Contrary to expectations, 20 of 22 twin spots exhibited aneuploidy or reciprocal translocations between non-homologous chromosomes. One twin spot showed a pattern consistent with a reciprocal crossover. Single white or green spots were [~]10X more frequent than twin spots. Among 26 single spots, 18 resulted from terminal arm deletion, 2 from aneuploidy, and 4 were associated with translocations. Deletion and reciprocal exchange breakpoints clustered near the centromere. Together, these findings indicate that nearly all spots arise from deletions, translocations, and other rearrangements likely mediated by chromosome missegregation or nonhomologous repair rather than by HR. These rearrangements altered the ratio of mutant to wild-type alleles and consequently chlorophyll levels. We conclude that mitotic recombination between homologous chromosomes is rare, and that genome instability is the dominant driver of somatic chimerism in this system.
Insulin-like growth factor 1 (IGF-1) is an anabolic signal promoting growth, differentiation and function of both embryonic and postnatal tissues. Both endocrine and paracrine functions of IGF-1 have been documented to regulate bone growth and bone marrow hematopoiesis. Local production of IGF-1 from various cell types may contribute differently to the overall bioactivity of IGF-1 in bone, but relevant sources and mechanisms are yet to be fully elucidated. Here we report that the Adipoq+ stromal cells are a notable source of IGF-1 in the bone marrow of postnatal mice. Deletion of IGF-1 with Adipoq-Cre diminished endocortical bone formation and cortical bone mass in mature female mice. On the other hand, the trabecular bone parameters or hematopoietic properties were not affected in mutant mice of either sex. The study uncovers a local source of IGF-1 in the bone marrow microenvironment that contributes to bone anabolic regulation in a site-specific manner. Lay SummaryThe insulin-like growth factor 1 (IGF-1) is known to play important roles in promoting bone growth and in regulating blood stem or progenitor cells. The factor is available both through the blood circulation and from the local production by cells in the bone tissue, but the functional contribution of each source is not well understood. Here we discover the non-blood cells in the bone marrow (bone marrow stromal cells) as a notable source of IGF-1 which specifically stimulates bone formation in the bone shaft of the long bone.
Fusobacterium nucleatum is a Gram-negative anaerobe associated with periodontitis and colorectal cancer. It secretes putrescine, a polyamine that promotes biofilm formation by oral co-colonizers and enhances the proliferation of cancer cells. However, the physiological importance of putrescine for F. nucleatum itself remains unexplored. Here, we show that putrescine biosynthesis, mediated by the ornithine decarboxylase gene oda, is essential for F. nucleatum viability. Deletion of oda was only possible when a functional copy was provided in trans, and CRISPR interference of oda expression resulted in complete growth arrest. The essentiality of oda was conserved across multiple subspecies. Supplementation with exogenous putrescine enabled the isolation of a conditional oda mutant whose growth was strictly putrescine-dependent. Putrescine depletion caused filamentation, membrane disruption, detergent hypersensitivity, and lysis in hypoosmotic conditions, indicating a critical role in maintaining cell envelope integrity. RNA sequencing revealed broad transcriptional remodeling under putrescine-limited conditions, including upregulation of genes involved in lipid metabolism, osmoprotection, and cell wall remodeling. Notably, oda transcript levels increased when putrescine was depleted, suggesting a negative feedback mechanism. These findings demonstrate that putrescine is not only an extracellular communal metabolite but is also vital for the cellular integrity and survival of F. nucleatum under anaerobic conditions. IMPORTANCEFusobacterium nucleatum is a prominent member of the oral microbiota and has been linked to various human diseases, including periodontitis, preterm birth, and colorectal cancer. Despite its clinical significance, the metabolic requirements that support its growth and viability remain poorly understood. In this study, we identify the oda gene, which encodes ornithine decarboxylase, as essential for F. nucleatum survival due to its role in putrescine biosynthesis. We demonstrate that depletion of putrescine leads to severe growth and morphological defects, accompanied by widespread transcriptional changes. These findings reveal an underappreciated metabolic vulnerability and highlight the critical role of polyamine homeostasis in maintaining cellular integrity in this notorious anaerobe.
Microglia are the resident immune cells of the central nervous system (CNS) and display diverse functions under both physiological and pathological conditions. The past decade has seen burgeoning interest in microglia function, with a variety of transgenic tools developed for specific genetic manipulation of microglia in various injury, disease, and developmental models. Although the majority of models have been developed in mice, the ability to manipulate microglia in rats provides additional advantages to studying microglial function in the brain especially related to complex behavior. Using BAC transgenesis, our lab has created a transgenic rat (Cx3cr1-CreERT2) that expresses a tamoxifen inducible Cre recombinase (CreERT2) under control of the microglial/macrophage specific fractalkine C-X3-C Motif Chemokine Receptor 1 (Cx3cr1) promoter. In mice, CreERT2 and other transgenes have been expressed in microglia using the Cx3cr1 promoter, however, this is the first demonstration in rats. Importantly, these rats exhibit similar cognitive behaviors compared to their wildtype (WT) controls. Microglial specificity of inducible Cre expression was confirmed by breeding the novel Cx3cr1-CreERT2+/- rat with a previously reported double floxed inverse open reading frame (DIO)-mCherry+/- reporter rat to show tamoxifen inducible mCherry expression that colocalizes with the microglial marker Iba1. In addition, we utilize flow cytometry to demonstrate time and Cre dependent differences in recombination of Cx3cr1+ cells in the spleen, peripheral blood, and brain at two- and eight-weeks post-tamoxifen treatment. Overall, we have created a novel transgenic rat model for researchers to employ in understanding microglial and peripheral immune cell function in rats.
Despite internal cues that signal fullness, animals can continue eating when motivated by context or palatability. The neural pathways and signals that enable animals to override these fullness cues remain unclear. We examined a central amygdala (CeA) projection to the dorsolateral pons that targets the parabrachial nucleus, a well-established meal termination center, and the adjacent supratrigeminal nucleus, a region that controls orofacial movements. Activity in this CeApons pathway correlated with the animals licking behavior but was not modulated by metabolic need or palatability cues. CeApons stimulation caused animals to overeat, consume non-edible objects within reach, or exhibit ingestion-like behaviors--licking, chewing, and grasping--even when no target was present. Depending on training and context, stimulation elicited either licking or pellet consumption, suggesting that CeApons promotes a flexible, goal-directed ingestive state by recruiting consummatory motor circuits rather than simply suppressing satiety signals. These findings highlight how forebrain-brainstem interactions can re-engage feeding behavior beyond homeostatic need.
There is little understanding of how aging serves as the strongest risk factor for several neurogenerative diseases. Specific neural cell types, such as microglia, undergo age-related maladaptive changes, including increased inflammation, impaired debris clearance, and cellular senescence, yet specific mediators that regulate these processes remain unclear. The aged brain is rejuvenated by youth-associated plasma factors, including tissue inhibitor of metalloproteinases 2 (TIMP2), which we have shown acts on the extracellular matrix (ECM) to regulate synaptic plasticity. Given emerging roles for microglia in these processes, we examined the impact of TIMP2 on microglial function. We show that TIMP2 deletion exacerbates microglial phenotypes associated with aging, including transcriptomic changes in cell activation, increased microgliosis, and increased levels of stress and inflammatory proteins measured in the brain extracellular space by in vivo microdialysis. Deleting specific cellular pools of TIMP2 in vivo increased microglial activation and altered myelin phagocytosis. Treating aged mice with TIMP2 reversed several phenotypes observed in our deletion models, resulting in decreased microglial activation, reduced proportions of proinflammatory microglia, and enhanced phagocytosis of physiological substrates. Our results identify TIMP2 as a key modulator of age-associated microglia dysfunction. Harnessing its activity may mitigate detrimental effects of age-associated insults on microglia function.
Many inflammatory stimuli can induce progenitor cells in the bone marrow to produce increased numbers of myeloid cells as part of the process of emergency myelopoiesis. These events are associated with trained immunity and have long-term impacts on hematopoietic stem and progenitor cell (HSPC) development but can also compromise their function. While many cytokines support emergency myelopoiesis, less is known about the mechanisms that temper these events. When mice that lack the cytokine IL-27 were infected with Toxoplasma gondii, there was enhanced generation of monocyte progenitors and increased numbers of inflammatory monocytes. In the bone marrow of infected mice there was increased production of IL-27 that localized with HSPCs and a survey of cytokine receptor expression highlighted that HSPCs were uniquely poised to respond to IL-27. Furthermore, the use of in vitro differentiation assays and mixed bone marrow chimeras revealed that HSPCs from IL-27 deficient mice are pre-disposed towards the monocyte lineage. Additional studies highlighted that after infection loss of the IL-27R resulted in reduced HSPC fitness that manifested as reduced proliferative responses and a decreased ability to reconstitute the hematopoietic system. Thus, the ability of IL-27 to act on HSPC provides a regulatory brake on differentiation to limit monocyte induction and preserve HSPC stemness.
While cannabis use during pregnancy is often perceived as harmless, little is known about its consequences on offspring neurodevelopment. There is an urgent need to map the effects of prenatal cannabis exposure on the brain through the course of the lifespan. We used magnetic resonance imaging spanning nine timepoints, behavioral assays, and electron microscopy to build a trajectory from gestation to adulthood in mice exposed prenatally to delta-9-tetrahydrocannabinol (THC). Our results demonstrate a spatio-temporal patterning, with ventriculomegaly in THC-exposed embryos followed by a deceleration of brain growth in neonates that is sustained until adulthood, especially in females. We observed consistently impacted regions in both the cortex and subcortex, aligned with sex-dependent changes to social behavior in neonates and increased anxiety-like behavior in adolescents. Our results suggest prenatal THC exposure has a sustained sex-dependent impact on neurodevelopment that may persist into early adulthood.
The resources required to study gene function are limited, especially when considering the number of genes in the human genome and the complexity of their function. Therefore, genes are prioritized for experimental studies based on many different considerations, including, but not limited to, perceived biomedical importance, such as disease-associated genes, or the understanding of biological processes, such as cell signaling pathways. At the same time, most genes are not studied or are under-characterized, which hampers our understanding of their function and potential effects on human health and wellness. Understanding function annotation disparity is a necessary first step toward understanding how much functional knowledge is gained from the human genome, and toward guidelines for better targeting future studies of the genes in the human genome effectively. Here, we present a comprehensive longitudinal analysis of the human proteome utilizing data analysis tools from economics and information theory. Specifically, we view the human proteome as a population of proteins within a knowledge economy: we treat the quantified knowledge of the proteins function as the analog of wealth and examine the distribution of information in a population of proteins in the proteome in the same manner distribution of wealth is studied in societies. Our results show a highly skewed distribution of information about human proteins over the last decade, in which the inequality in the annotations given to the proteins remains high. Additionally, we examine the correlation between the knowledge about protein function as captured in databases and the interest in proteins as reflected by mentions in the scientific literature. We show a large gap between knowledge and interest and dissect the factors leading to this gap. In conclusion, our study shows that research efforts should be redirected to less studied proteins to mitigate the disparity among human proteins both in databases and literature.
The epithelial to mesenchymal transition (EMT) is a widely studied but poorly defined state change due to the variety of ways in which it has been characterized in cells. There is a need for reproducible cell model systems that enable the integration and comparison of different types of measured observations of cells across many distinct cellular contexts. We present human induced pluripotent stem (hiPS) cells as such a model system by demonstrating its utility through a comparative analysis of hiPS cell-EMT in 2D and 3D cell culture geometries. We developed live-imaging-based assays to directly compare examples of changes in cell function (via migration timing), molecular components (via expression of marker proteins), organization (via reorganization of cell junctions), and environment (via dynamics of basement membrane) in the same experimental system. The EMT-related changes we measured occurred earlier in 2D colonies than in 3D lumenoids, likely due to differences in the basement membrane environments associated with 2D vs. 3D initial hiPS cell culture geometries. We have made the 449 60-hour-long 3D time-lapse movies and the associated tools used for analysis and visualization open-source and easily accessible as a resource for future work in this field.
BackgroundGiven the resurgence of syphilis, research endeavors to improve current assays for serological diagnosis and management of this disease are a priority. A proteome-scale platform for high-throughput profiling of the humoral response to Treponema pallidum (T. pallidum) proteins during infection could identify antigens suitable to ameliorate the performance and capabilities of treponemal tests (TTs), which may require weeks to become positive following infection, cannot distinguish between active and previously treated infections, or assess treatment response. Additionally, because infection-induced immunity is partially protective, profiling the response to T. pallidum outer membrane proteins (OMPs) could help select vaccine candidates. MethodsWe developed a pan-proteome array (PPA) based on the Nichols and SS14 strain complete proteomes and used it to define the IgM and IgG humoral response to 1,009 T. pallidum proteins in sera collected longitudinally from long-term infected rabbits, and from rabbits that were infected, treated, and re-infected. FindingsApproximately a third of the pathogens proteome was recognized in infected animals, with a marked IgG response detectable between day-10 and day-20 post-infection. We found early, gradual, and late IgG kinetic profiles, strain-dependent differences in humoral reactivity, and post-treatment fluctuation in reactivity for several antigens. Very few antigens elicited an IgM response. Several OMPs were significantly and differentially recognized, but few elicited a robust response. InterpretationThe PPA allowed the identification of antigens that could facilitate early diagnosis and of a core set of OMP that could explain protection upon re-infection. No antigen appeared suitable to monitor treatment response. FundingNIH SBIR-R43AI149804 RESEARCH IN CONTEXTO_ST_ABSEvidence before this studyC_ST_ABSIn April 2024, we searched the PubMed database for articles on preclinical studies using high throughput proteome arrays containing at least 10% of the predicted T. pallidum proteome that aimed at identifying antibody reactivity to T. pallidum antigens during experimental syphilis infection. We could retrieve only one manuscript. In this work, an array containing the T. pallidum partial proteome as annotated in the first sequenced Nichols strain genome (GCA_000008605.1) in 1998 was assembled using recombinant antigens expressed in Escherichia coli (E. coli). The resulting array was probed using pooled sera from three rabbits infected with the Nichols stain of T. pallidum, attained from infected animals at five time points following intratesticular infection. The small number of reactive antigens (n = 106) identified in this early study was likely to be an incomplete set of all antigens recognized during infection because not all the predicted targets in the T. pallidum proteome were successfully expressed and tested. In retrospect, additional limitations of the study included an initial suboptimal annotation of the Nichols genome used to define the pathogens proteome, which has now changed with the availability of a re-sequenced Nichols strain genome devoid of sequencing errors that affected the initial annotation process, and the refinement of bioinformatic pipelines for the identification of open reading frames (ORFs). Furthermore (as acknowledged by the authors), the possible presence of amplification errors in their expression clones might have affected the sequence of some protein targets and antibody binding to the targets. As a result, some of the T. pallidum antigens known to elicit a robust humoral response during experimental infection were not detected in this antigenic screen. Lastly, employing only the Nichols strain in this early study did not consider that a significant portion of the circulating syphilis strains belong to the SS14 clade of T. pallidum. Added value of this studyThis novel PPA, combined with a more robust experiential design than ever reported, allowed us to overcome most of the limitations associated with the study mentioned above, as we were able to a) use the most recent annotations for the selected T. pallidum strains based on accurate genome sequences, b) print the pathogens virtually complete proteome in the study array, c) analyze individual sera to account for rabbit-to-rabbit variability in the humoral response to infection rather than pooled sera, d) detect both IgM and IgG over 10 or 20 timepoints, depending on the experimental design, e) obtain information on how the humoral response evolved upon treatment and re-infection and, finally, f) evaluate all of the above in animals infected with two T. pallidum strains whose genetic background is representative of the two currently circulating clades of the syphilis agent. Implications of all the available evidenceOur study provides new and more comprehensive data on how humoral immunity for two classes of antibodies develops during infection and how it evolves in response to treatment and re-infection. The analysis of sera collected at tightly spaced time points post-inoculation and for an extensive period post-infection provides a wealth of information to improve the diagnostic performance of existing tests detecting treponemal antigens. The analysis of differential immunity specific to the pathogens putative OMPs provides a rationale for vaccine candidate selection.
Pseudomonas aeruginosa is a multidrug-resistant opportunistic human pathogen that can survive in many natural and anthropogenic environments. It is a leading cause of morbidity in individuals with cystic fibrosis and is one of the most prevalent pathogens associated with nosocomial infections in the United States. It has been shown that this organism can survive and persist in low nutrient environments, such as sink drains. How adaptation to these types of environments influences the phenotypic traits of this organism has not been well studied. Here we implemented an experimental evolution system in which six strains of P. aeruginosa were subjected to low nutrient conditions over the course of 12-weeks and assessed phenotypic and genotypic changes that occurred as a result of adaptation to such environments. We observed that adaptation to low nutrient environments resulted in decreased generation time, reduced cell size, reduced biofilm formation, increased pyocyanin production, and decreased motility for some of the strains. Further, some of the evolved isolates were significantly more virulent/competitive against a phagocytic predator. This study is significant as it allows us to predict how this organism will evolve in hospital and domestic environments and can help us improve treatment options for patients. ImportanceHuman commensal and pathogenic organisms undergo dynamic cycles across human and non-human environments. Despite the crucial implications for human health, the understanding of bacterial adaptations to these diverse environments and their subsequent impact on human-bacterial interactions remains underexplored. This study shows how P. aeruginosa, an opportunistic human pathogen, adapts phenotypically in response to a shift from high nutrients (like those found in the human body) to low nutrients (like those found in many other environments, like sink drains). This work also shows that, in some cases, resistance to predatory forces can evolve in the absence of a predator. This work is important as it contributes to the growing body of knowledge concerning how external, non-host-related abiotic conditions influence host-pathogen interactions.
The upper plant body is formed by the continued growth of shoot apical meristems. In angiosperms, meristems are organized in three cell layers that tend to remain clonally isolated. Somatic variants emerge when mutant cells overtake part or all of a meristem. During sexual reproduction, only one layer, the L2, contributes to the next generation by forming gametes. The L2 is known to frequently invade and replace the L3, while L1-L2 separation is persistent. The role of different meristem layers in mutation accumulation is unknown. We discovered a potato periclinal chimera in which the L2 and L3, but not the L1, are marked by a chromosomal translocation. This enabled the identification of plants regenerated from leaf protoplasts originating from either the L1 or L2+L3. Leveraging these layer-specific clones, we identified and compared mutations that accumulated in the layers in the clonal parent for several decades. Here we show that the L1 accumulates mutations at 1.9 times the rate of the L2, indicating that plants might protect the germline by mechanisms that reduce the rate of mutation accumulation in the L2. In contrast to these layer-specific mutations, we found no evidence of somatic mutation fixation in all three meristem layers. Our findings highlight how periclinal chimeras are formed by independent mutational processes in which a mutation-prone epidermal layer could increase clonal variation.
Genome-wide expression data has become ubiquitous within the last two decades. Given such data, functional enrichment methods identify functional categories (e.g., biological processes) that preferentially annotate differentially expressed genes. However, many existing methods operate in a binary manner, disregarding valuable information contained in the gene ranking. The few methods that consider the ranking often return redundant or non-specific functional categories. To address these limitations, we developed a novel method called Concise Ranked Functional Enrichment (CRFE), which effectively leverages the ranking information in gene expression data to compute a non-redundant set of specific functional categories that are notably enriched for highly ranked genes. A particularly useful feature of CRFE is a tunable parameter that defines how much focus should be given to the most highly ranked genes. Using four treatment-control RNA-seq datasets, we compared the performance of CRFE with the two most widely used types of functional enrichment methods, Gene Set Enrichment Analysis and over-representation analysis. We evaluated the methods based on their ability to utilize ranking information, generate non-redundant results, and return functional categories with high information content. CRFE excelled in all evaluated criteria, outperforming the existing methods, each of which exhibits deficiencies in at least one aspect. Using lung adenocarcinoma data, we further showed that the functional categories identified by CRFE are biologically meaningful. In conclusion, CRFE computes an informative set of functional categories that summarizes genome-wide expression data. With its superior performance over existing methods, CRFE harbors great promise to become a widely used functional enrichment method. Author summaryGiven a list of differentially expressed genes as input, functional enrichment methods reveal which functional categories (e.g., biological processes) were likely activated by the cell and are responsible for the differential expression. We developed a new such method, called Concise Ranked Functional Enrichment (CRFE), which addresses the limitations of current approaches by incorporating gene ranking information to compute a concise and specific set of enriched functional categories. Using four treatment-control RNA-seq datasets, we evaluate how well CRFE and the two currently most widely used methods perform in three criteria. We find that CRFE outperforms each of the alternative methods in at least one of the evaluated criteria, demonstrating its superiority. A high-level interpretation of the functional categories identified by CRFE for lung adenocarcinoma datasets highlights its usefulness for experimentalists. Overall, CRFE harnesses the power of ranked gene lists to generate a focused and non-redundant set of enriched functional categories. Our study positions CRFE as a promising method for functional enrichment analysis, with the potential to advance research in this field.
To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.