Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.

Advanced maternal age of first time pregnant mothers is associated with prolonged and dysfunctional labor and significant risk of emergency cesarean section. We investigated the influence of maternal age on myometrial contractility, expression of contractile associated proteins (CAPs), and global gene expression in the parturient uterus. Female Wistar rats either 8 (YOUNG n = 10) or 24 (OLDER n = 10) weeks old were fed laboratory chow, mated, and killed during parturition. Myometrial strips were dissected to determine contractile activity, cholesterol (CHOL) and triglycerides (TAG) content, protein expression of connexin-43 (GJA1), prostaglandin-endoperoxide synthase 2 (PTGS2), and caveolin 1 (CAV-1). Maternal plasma concentrations of prostaglandins PGE2, PGF2α, and progesterone were determined by RIA. Global gene expression in uterine samples was compared using Affymetrix Genechip Gene 2.0 ST arrays and Ingenuity Pathway analysis (IPA). Spontaneous contractility in myometrium exhibited by YOUNG rats was threefold greater than OLDER animals (P < 0.027) but maternal age had no significant effect on myometrial CAP expression, lipid profiles, or pregnancy-related hormones. OLDER myometrium increased contractile activity in response to PGF2α, phenylephrine, and carbachol, a response absent in YOUNG rats (all P < 0.002). Microarray analysis identified that maternal age affected expression of genes related to immune and inflammatory responses, lipid transport and metabolism, steroid metabolism, tissue remodeling, and smooth muscle contraction. In conclusion YOUNG laboring rat myometrium seems primed to contract maximally, whereas activity is blunted in OLDER animals and requires stimulation to meet contractile potential. Further work investigating maternal age effects on myometrial function is required with focus on lipid metabolism and inflammatory pathways.

The role of h1-calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1-calponin in their smooth muscles. The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild-type (WT) mice, nor were the delays in onset and half-times of Ca2+ sensitization, initiated by flash photolysis of caged GTPgammaS, different. The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P<0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of Vus was proportional to its actomyosin binding in situ. Actin expression was reduced by 25-50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca2+-sensitization pathways in smooth muscle.

Molecular mechanisms underlying bladder dysfunction in ischemia, particularly at the protein and protein modification levels and downstream pathways, remain largely unknown. Here we describe a comparison of protein sequence variations in the ischemic and normal bladder tissues by measuring the mass differences of the coding amino acids and actual residues crossing the proteome. A large number of nonzero delta masses (11,056) were detected, spanning over 1295 protein residues. Clustering analysis identified 12 delta mass clusters that were significantly dysregulated, involving 30 upregulated (R2 > 0.5, ratio > 2, p < 0.05) and 33 downregulated (R2 > 0.5, ratio < -2, p < 0.05) proteins in bladder ischemia. These protein residues had different mass weights from those of the standard coding amino acids, suggesting the formation of non-coded amino acid (ncAA) residues in bladder ischemia. Pathway, gene ontology, and protein-protein interaction network analyses of these ischemia-associated delta-mass containing proteins indicated that ischemia provoked several amino acid variations, potentially post-translational modifications, in the contractile proteins and stress response molecules in the bladder. Accumulation of ncAAs may be a novel biomarker of smooth muscle dysfunction, with diagnostic potential for bladder dysfunction. Our data suggest that systematic assessment of global protein modifications may be crucial to the characterization of ischemic conditions in general and the pathomechanism of bladder dysfunction in ischemia.

Urinary bladder smooth muscle contraction is triggered by parasympathetic nerves, which release ATP and acetylcholine (ACh) that bind to purinergic and muscarinic receptors, respectively. Neuronal signalling may thus elicit myosin regulatory light chain (RLC) phosphorylation and contraction through the combined, but distinct contributions of these receptors. Both receptors mediate Ca2+ influx whereas muscarinic receptors may also recruit Ca2+-sensitization mechanisms. Using transgenic mice expressing calmodulin sensor myosin light chain kinase (MLCK) in smooth muscles, the effects of suramin/α,β-methylene ATP (α,β-meATP) (purinergic inhibition) or atropine (muscarinic inhibition) on neurally stimulated elevation of [Ca2+]i, MLCK activation, force and phosphorylation of RLC, myosin light chain phosphatase (MLCP) targeting subunit MYPT1 and MLCP inhibitor protein CPI-17 were examined. Electric field stimulation (EFS) increased [Ca2+]i, MLCK activation and concomitant force in a frequency-dependent manner. The dependence of force on [Ca2+]i and MLCK activation decreased with time suggesting increased Ca2+ sensitization in the late contractile phase. RLC and CPI-17 phosphorylation increased upon stimulation with maximal responses at 20 Hz; both responses were attenuated by atropine, but only RLC phosphorylation was inhibited by suramin/α,β-meATP. Antagonism of purinergic receptors suppressed maximal MLCK activation to a greater extent in the early contractile phase than in the late contractile phase; atropine had the opposite effect. A frequency- and time-dependent increase in MLCK phosphorylation explained the desensitization of MLCK to Ca2+, since MLCK activation declined more rapidly than [Ca2+]i. EFS elicited little or no effect on MYPT1 Thr696 or 850 phosphorylation. Thus, purinergic Ca2+ signals provide the initial activation of MLCK with muscarinic receptors supporting sustained responses. Activation of muscarinic receptors recruits CPI-17, but not MYPT1-mediated Ca2+ sensitization. Furthermore, nerve-released ACh also initiates signalling cascades leading to phosphorylation-dependent desensitization of MLCK.

Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140 kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.

The active and passive contractile performance of skeletal muscle fibers largely depends on the myosin heavy chain (MHC) isoform and the stiffness of the titin spring, respectively. Open questions concern the relationship between titin-based stiffness and active contractile parameters, and titin's importance for total passive muscle stiffness. Here, a large set of adult rabbit muscles (n = 37) was studied for titin size diversity, passive mechanical properties, and possible correlations with the fiber/MHC composition. Titin isoform analyses showed sizes between approximately 3300 and 3700 kD; 31 muscles contained a single isoform, six muscles coexpressed two isoforms, including the psoas, where individual fibers expressed similar isoform ratios of 30:70 (3.4:3.3 MD). Gel electrophoresis and Western blotting of two other giant muscle proteins, nebulin and obscurin, demonstrated muscle type-dependent size differences of < or =70 kD. Single fiber and single myofibril mechanics performed on a subset of muscles showed inverse relationships between titin size and titin-borne tension. Force measurements on muscle strips suggested that titin-based stiffness is not correlated with total passive stiffness, which is largely determined also by extramyofibrillar structures, particularly collagen. Some muscles have low titin-based stiffness but high total passive stiffness, whereas the opposite is true for other muscles. Plots of titin size versus percentage of fiber type or MHC isoform (I-IIB-IIA-IID) determined by myofibrillar ATPase staining and gel electrophoresis revealed modest correlations with the type I fiber and MHC-I proportions. No relationships were found with the proportions of the different type II fiber/MHC-II subtypes. Titin-based stiffness decreased with the slow fiber/MHC percentage, whereas neither extramyofibrillar nor total passive stiffness depended on the fiber/MHC composition. In conclusion, a low correlation exists between the active and passive mechanical properties of skeletal muscle fibers. Slow muscles usually express long titin(s), predominantly fast muscles can express either short or long titin(s), giving rise to low titin-based stiffness in slow muscles and highly variable stiffness in fast muscles. Titin contributes substantially to total passive stiffness, but this contribution varies greatly among muscles.

Maternal obesity is associated with prolonged and dysfunctional labour and emergency caesarean section, but the mechanisms are unknown. The present study investigated the effects of an adiposity-inducing high-fat, high-cholesterol (HFHC) diet on uterine contractile-associated protein (CAP) expression and ex vivo uterine contractility in term non-labouring (TNL) and term labouring (TL) rats. Female rats were fed either control chow (CON n=20) or HFHC (n=20) diet 6 weeks before conception and during pregnancy. On gestational day 21 (TNL) or day 22 (TL) CON and HFHC (n=10) rats were killed to determine plasma cholesterol, triacylglycerol and progesterone concentrations and collection of myometrium for contractility studies and expression of CAPs caveolin-1 (Cav-1), connexin-43 (CX-43) and it's phosphorylated form (pCX-43), oxytocin receptor (OXTR) and cyclooxygenase-2 (COX-2). HFHC feeding increased visceral fat (P≤0.001), plasma cholesterol (P≤0.001) and triacylglycerol (P=0.039) concentrations. Stage of labour effected uterine expression of CAV-1 (P<0.02), pCX43 and COX-2 (both P<0.03). CAV-1 and pCX43 decreased but COX-2 increased with parturition. Significant diet- and labour-stage interactions were evident for CX-43 and pCX43 (P<0.03 and P<0.004 respectively). CX-43 decreased with TL in HFHC animals but was unaltered in CON. pCX-43 fell with labour in CON but remained high in HFHC. OXTR expression was significantly higher in HFHC compared with CON animals (P<0.03). Progesterone was higher in HFHC rats at term (P<0.014) but fell significantly with labour to similar concentrations as CON. Contractility studies identified synchronous contractions of stable amplitude in lean animals, but unstable asynchronous contractions with obesity. Uterine dose response to oxytocin was blunted during labour in HFHC rats with a log EC50 of -8.84 compared with -10.25 M in CON for integral activity (P<0.05). In conclusion, our adiposity model exhibits adverse effects on contractile activity during labour that can be investigated further to unravel the mechanisms causing uterine dystocia in obese women.

Heme released from red blood cells targets a number of cell components including the cytoskeleton. The purpose of the present study was to determine the impact of free heme (20-300 µM) on human skeletal muscle fibres made available during orthopedic surgery. Isometric force production and oxidative protein modifications were monitored in permeabilized skeletal muscle fibre segments. A single heme exposure (20 µM) to muscle fibres decreased Ca2+-activated maximal (active) force (Fo) by about 50% and evoked an approximately 3-fold increase in Ca2+-independent (passive) force (Fpassive). Oxidation of sulfhydryl (SH) groups was detected in structural proteins (e.g., nebulin, α-actinin, meromyosin 2) and in contractile proteins (e.g., myosin heavy chain and myosin-binding protein C) as well as in titin in the presence of 300 µM heme. This SH oxidation was not reversed by dithiothreitol (50 mM). Sulfenic acid (SOH) formation was also detected in the structural proteins (nebulin, α-actinin, meromyosin). Heme effects on SH oxidation and SOH formation were prevented by hemopexin (Hpx) and α1-microglobulin (A1M). These data suggest that free heme has a significant impact on human skeletal muscle fibres, whereby oxidative alterations in structural and contractile proteins limit contractile function. This may explain and or contribute to the weakness and increase of skeletal muscle stiffness in chronic heart failure, rhabdomyolysis, and other hemolytic diseases. Therefore, therapeutic use of Hpx and A1M supplementation might be effective in preventing heme-induced skeletal muscle alterations.

Chronic airway diseases, like asthma or COPD, are characterized by excessive acetylcholine release and airway remodeling. The aim of this study was to investigate the long-term effect of muscarinic agonists on the phenotype and proliferation of rabbit tracheal airway smooth muscle cells (ASMCs).

To establish a reliable isoproterenol induced heart dysfunction fetal rat model and understand the switches of contractile proteins, 45 pregnant rats were divided into 15 mg/kg-once, 15 mg/kg-twice, sham-operated once, sham-operated twice, and control groups. And 18 adult rats were divided into isoproterenol-treated and control groups. H&E staining, Masson staining, and transmission electron microscope were performed. Apoptotic rate assessed by TUNEL analysis and expressions of ANP, BNP, MMP-2, and CTGF of hearts were measured. Intra-amniotic injections of isoproterenol were supplied on E14.5 and E15.5 for fetuses and 7-day continuous intraperitoneal injections were performed for adults. Then echocardiography was performed with M-mode view assessment on E18.5 and 6 weeks later, respectively. Isoproterenol twice treated fetuses exhibited significant changes in histological evaluation, and mitochondrial damages were significantly severe with increased apoptotic rate. ANP and BNP increased and that of MMP-2 increased in isoproterenol twice treated group compared to control group, without CTGF. The isoforms transition of troponin I and myosin heavy chain of fetal heart dysfunction were opposite to adult procedure. The administration of intra-amniotic isoproterenol to fetal rats could induce heart dysfunction and the regulation of contractile proteins of fetuses was different from adult procedure.

While the long-term physiological adaptation of the neuromuscular system to changed functional demands is usually reflected by unilateral skeletal muscle transitions, the progressive degeneration of distinct motor neuron populations is often associated with more complex changes in the abundance and/or isoform expression pattern of contractile proteins and metabolic enzymes. In order to evaluate these intricate effects of primary motor neuronopathy on the skeletal muscle proteome, label-free MS was employed to study global alterations in the WR (wobbler) mouse model of progressive neurodegeneration. In motor neuron disease, fibre-type specification and the metabolic weighting of bioenergetic pathways appear to be strongly influenced by both a differing degree of a subtype-specific vulnerability of neuromuscular synapses and compensatory mechanisms of fibre-type shifting. Proteomic profiling confirmed this pathobiochemical complexity of disease-induced changes and showed distinct alterations in 72 protein species, including a variety of fibre-type-specific isoforms of contractile proteins, metabolic enzymes, metabolite transporters and ion-regulatory proteins, as well as changes in molecular chaperones and various structural proteins. Increases in slow myosin light chains and the troponin complex and a decrease in fast MBP (myosin-binding protein) probably reflect the initial preferential loss of the fast type of neuromuscular synapses in motor neuron disease.

Coronavirus disease 2019 (COVID-19) is associated with adverse impacts in the cardiovascular system, but the mechanisms driving this response remain unclear. In this study, we conducted "pseudoviral infection" of SARS-CoV-2 subunits to evaluate their toxic effects in cardiomyocytes (CMs), that were derived from human induced pluripotent stem cells (hiPSCs). We found that the ectopic expression of S and ORF-9B subunits significantly impaired the contractile function and altered the metabolic profiles in human cardiomyocytes. Further mechanistic study has shown that the mitochondrial oxidative phosphorylation (OXPHOS), membrane potential, and ATP production were significantly decreased two days after the overexpression of S and ORF-9B subunits, while S subunits induced higher level of reactive oxygen species (ROS). Two weeks after overexpression, glycolysis was elevated in the ORF-9B group. Based on the transcriptomic analysis, both S and ORF-9B subunits dysregulated signaling pathways associated with metabolism and cardiomyopathy, including upregulated genes involved in HIF-signaling and downregulated genes involved in cholesterol biosynthetic processes. The ORF-9B subunit also enhanced glycolysis in the CMs. Our results collectively provide an insight into the molecular mechanisms underlying SARS-CoV-2 subunits-induced metabolic alterations and cardiac dysfunctions in the hearts of COVID-19 patients.

The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetal- and adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro Tissues (CMT) and Tissue Strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca2+-handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simultaneous SMYD1 overexpression and SMARCD1 suppression in hESC-VCMs synergistically interacted to increase the contractile forces of CMTs and CTSs with up-regulated transcripts for cardiac contractile, Ca2+-handing, and ion channel proteins. Certain effects that were not detected at the single-cell level could be unleashed under 3D environments. The two chromatin remodelers SMYD1 and SMARCD1 play distinct roles in cardiac development and maturation, consistent with the notion that epigenetic priming requires triggering signals such as 3D environmental cues for pro-maturation effects.

Vascular endothelial zinc finger 1 (Vezf1) is a transcription factor previously shown to regulate vasculogenesis and angiogenesis. We aimed to investigate the role of Vezf1 in the postnatal heart.

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0-80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80-160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160-350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Contractile actin cortex is involved in cell morphogenesis, movement, and cytokinesis, but its organization and assembly are poorly understood. During blebbing, the membrane detaches from the cortex and inflates. As expansion ceases, contractile cortex re-assembles under the membrane and drives bleb retraction. This cycle enabled us to measure the temporal sequence of protein recruitment to the membrane during cortex reassembly and to explore dependency relationships. Expanding blebs were devoid of actin, but proteins of the erythrocytic submembranous cytoskeleton were present. When expansion ceased, ezrin was recruited to the membrane first, followed by actin, actin-bundling proteins, and, finally, contractile proteins. Complete assembly of the contractile cortex, which was organized into a cagelike mesh of filaments, took approximately 30 s. Cytochalasin D blocked recruitment of actin and alpha-actinin, but had no effect on membrane association of ankyrin B and ezrin. Ezrin played no role in actin nucleation, but was essential for tethering the membrane to the cortex. The Rho pathway was important for cortex assembly in blebs.

Siphoviruses are main killers of bacteria. They use a long non-contractile tail to recognize the host cell and to deliver the genome from the viral capsid to the bacterial cytoplasm. Here, we define the molecular organization of the Bacillus subtilis bacteriophage SPP1 ~ 6.8 MDa tail and uncover its biogenesis mechanisms. A complex between gp21 and the tail distal protein (Dit) gp19.1 is assembled first to build the tail cap (gp19.1-gp21Nter) connected by a flexible hinge to the tail fiber (gp21Cter). The tip of the gp21Cter fiber is loosely associated to gp22. The cap provides a platform where tail tube proteins (TTPs) initiate polymerization around the tape measure protein gp18 (TMP), a reaction dependent on the non-structural tail assembly chaperones gp17.5 and gp17.5* (TACs). Gp17.5 is essential for stability of gp18 in the cell. Helical polymerization stops at a precise tube length followed by binding of proteins gp16.1 (TCP) and gp17 (THJP) to build the tail interface for attachment to the capsid portal system. This finding uncovers the function of the extensively conserved gp16.1-homologs in assembly of long tails. All SPP1 tail components, apart from gp22, share homology to conserved proteins whose coding genes' synteny is broadly maintained in siphoviruses. They conceivably represent the minimal essential protein set necessary to build functional long tails. Proteins homologous to SPP1 tail building blocks feature a variety of add-on modules that diversify extensively the tail core structure, expanding its capability to bind host cells and to deliver the viral genome to the bacterial cytoplasm.

Contractile injection systems (CIS) are bacteriophage tail-like structures that mediate bacterial cell-cell interactions. While CIS are highly abundant across diverse bacterial phyla, representative gene clusters in Gram-positive organisms remain poorly studied. Here we characterize a CIS in the Gram-positive multicellular model organism Streptomyces coelicolor and show that, in contrast to most other CIS, S. coelicolor CIS (CISSc) mediate cell death in response to stress and impact cellular development. CISSc are expressed in the cytoplasm of vegetative hyphae and are not released into the medium. Our cryo-electron microscopy structure enabled the engineering of non-contractile and fluorescently tagged CISSc assemblies. Cryo-electron tomography showed that CISSc contraction is linked to reduced cellular integrity. Fluorescence light microscopy furthermore revealed that functional CISSc mediate cell death upon encountering different types of stress. The absence of functional CISSc had an impact on hyphal differentiation and secondary metabolite production. Finally, we identified three putative effector proteins, which when absent, phenocopied other CISSc mutants. Our results provide new functional insights into CIS in Gram-positive organisms and a framework for studying novel intracellular roles, including regulated cell death and life-cycle progression in multicellular bacteria.