Phagosome Transport Research Articles

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins -1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein-complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

Acidification of the phagosome orchestrates the motor forces directing its transport

Phagosomes are dynamic organelles formed by macrophages to capture and destroy microbial pathogens. Phagosome transport from the cell periphery to the perinuclear region, is essential for fusion with lysosomes and the elimination of pathogens. Molecular motors, kinesin and dynein, generate opposing forces, transporting the phagosome away from and towards the lysosome, respectively. Luminal acidification plays a crucial role in determining the net directional movement of the phagosome. The mechanics of this regulation are not known. In this study, we used the sodium proton exchanger NHE9 to selectively modulate phagosomal acidification in macrophages. We then investigated its impact on the mechanical properties of kinesin and dynein motors through optical trapping experiments. We observed a negative correlation between the tenacity of dynein motors and pH under high resistive forces. Reduced luminal acidification impaired generation of dynein cooperative forces, which are crucial for transporting the phagosome to the lysosome. Conversely, the kinesin-powered motility of phagosomes is enabled by a decrease in phagosomal acidification. Given the various methods pathogens employ to limit phagosomal acidification, our findings are highly significant in the context of host-pathogen interactions.

Timing of Phagosome Maturation Depends on Their Transport Switching from Actin to Microtubule Tracks.

Phagosomes, specialized membrane compartments responsible for digesting internalized pathogens, undergo sequential dynamic and biochemical changes as they mature from nascent phagosomes to degradative phagolysosomes. Maturation of phagosomes depends on their transport along actin filaments and microtubules. However, the specific quantitative relationship between the biochemical transformation and transport dynamics remains poorly characterized. The autonomous nature of phagosomes, moving and maturing at different rates, makes understanding this relationship challenging. Addressing this challenge, in this study we engineered particle sensors to image and quantify single phagosomes' maturation. We found that as phagosomes move from the actin cortex to microtubule tracks, the timing of their actin-to-microtubule transition governs the duration of the early phagosome stage before acquiring degradative capacities. Prolonged entrapment of phagosomes in the actin cortex extends the early phagosome stage by delaying the dissociation of early endosome markers and phagosome acidification. Conversely, a shortened transition from actin- to microtubule-based movements causes the opposite effect on phagosome maturation. These results suggest that the actin- and microtubule-based transport of phagosomes functions like a "clock" to coordinate the timing of biochemical events during phagosome maturation, which is crucial for effective pathogen degradation.

The phagosomal solute transporter SLC15A4 promotes inflammasome activity via mTORC1 signaling and autophagy restraint in dendritic cells.

Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1β production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1β production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1β levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.

Characterization of Global Gene Expression, Regulation of Metal Ions, and Infection Outcomes in Immune-Competent 129S6 Mouse Macrophages.

Nutritional immunity involves cellular and physiological responses to invading pathogens, such as limiting iron, increasing exposure to bactericidal copper, and altering zinc to restrict the growth of pathogens. Here, we examine infection of bone marrow-derived macrophages from 129S6/SvEvTac mice by Salmonella enterica serovar Typhimurium. The 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations that plays an important role in modulating metal availability to the pathogen. We carried out global RNA sequencing upon treatment with live or heat-killed Salmonella at 2 h and 18 h postinfection and observed widespread changes in metal transport, metal-dependent genes, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells. Changes in host cell gene expression suggest infection increases cytosolic zinc while simultaneously limiting zinc within the phagosome. Using a genetically encoded sensor, we demonstrate that cytosolic labile zinc increases 45-fold at 12 h postinfection. Further, manipulation of zinc in the medium alters bacterial clearance and replication, with zinc depletion inhibiting both processes. Comparing the transcriptomic changes to published data on infection of C57BL/6 macrophages revealed notable differences in metal regulation and the global immune response. Our results reveal that 129S6 macrophages represent a distinct model system compared to C57BL/6 macrophages. Further, our results indicate that manipulation of zinc at the host-pathogen interface is more nuanced than that of iron or copper. The 129S6 macrophages leverage intricate means of manipulating zinc availability and distribution to limit the pathogen's access to zinc, while simultaneously ensuring sufficient zinc to support the immune response.

Deletion Mutants of Francisella Phagosomal Transporters FptA and FptF Are Highly Attenuated for Virulence and Are Protective Against Lethal Intranasal Francisella LVS Challenge in a Murine Model of Respiratory Tularemia.

Francisella tularensis (Ft) is a Gram-negative, facultative intracellular bacterium that is a Tier 1 Select Agent of concern for biodefense for which there is no licensed vaccine. A subfamily of 9 Francisella phagosomal transporter (fpt) genes belonging to the Major Facilitator Superfamily of transporters was identified as critical to pathogenesis and potential targets for attenuation and vaccine development. We evaluated the attenuation and protective capacity of LVS derivatives with deletions of the fptA and fptF genes in the C57BL/6J mouse model of respiratory tularemia. LVSΔfptA and LVSΔfptF were highly attenuated with LD50 values of >20 times that of LVS when administered intranasally and conferred 100% protection against lethal challenge. Immune responses to the fpt mutant strains in mouse lungs on day 6 post-infection were substantially modified compared to LVS and were associated with reduced organ burdens and reduced pathology. The immune responses to LVSΔfptA and LVSΔfptF were characterized by decreased levels of IL-10 and IL-1β in the BALF versus LVS, and increased numbers of B cells, αβ and γδ T cells, NK cells, and DCs versus LVS. These results support a fundamental requirement for FptA and FptF in the pathogenesis of Ft and the modulation of the host immune response.

Simultaneous Imaging of Dynamics and Biochemical Activities of Single Phagosomes

Phagocytosis is a major defense mechanism used by our immune system to eliminate pathogens. It proceeds in multiple steps: ligand-receptor recognition between pathogens and cell membranes in the beginning, internalization of pathogens into intracellular membrane organelles known as phagosomes, transport of phagosomes from cell edge to areas surrounding the cell nucleus, and digestion of the encapsulated pathogens through series of chemical reactions. All those steps must occur in the right order at the right time and in the right subcellular location. Abnormalities in any of those steps can lead to progression of infections and other serious human diseases, such as cystic fibrosis and chronic granulomatous disease. Thus, a fundamental understanding of phagocytosis is critical for developing methods to diagnose and treat those diseases. Unfortunately, that has been a grand challenging due to the complexity of the mechanisms involved in phagocytosis. To address the critical need, we developed nanoparticle-based sensors to probe the chemical and dynamical activities during phagocytosis. Simultaneous measurements of phagosome transport, acidification, and fusion reveal a striking correlation between the intracellular transport of phagosomes and their biochemical activities during maturation. Our results reveal a regulatory mechanism underlying the orchestration of dynamical and biochemical signaling processes during phagosome maturation.

Kinetic Separation of Oxidative and Non-oxidative Metabolism in Single Phagosomes from Alveolar Macrophages: Impact on Bacterial Killing.

SummaryThe relative contribution of the two phagosomal catabolic processes, oxidative and metabolic, was assessed in the killing of Pseudomonas aeruginosa in phagosomes of alveolar macrophages (AMs) from wild-type (p47-phox+/+) or NOX-defective (p47-phox−/−) mice. Free radical release and degradative acidification within AM phagosomes is sequential and separable. The initial NOX activity, identifiable as a transient alkalinization, leads to fast bacterial wall permeabilization by ROS. This is followed by V-ATPase-induced acidification and enzymatic bacterial degradation contributed through phagosomal-lysosomal fusion. The alkalinization/acidification ratio was variable among phagosomes within single cells of a given genotype and not as a function of macrophage M1 or M2 classification, possibly owing to uneven distribution of phagosomal transporter proteins. Irregular, excessive NOX activity prevents phago-lysosomal fusion, and the lack of V-ATPase-induced acidification leads to bacterial stasis in the phagosome. Thus, efficient phagosomal bacterial killing is a result of tightly balanced activity between two processes.

Physical Determinants of Particle Uptake and Transport during Phagocytosis

Phagocytosis is a central part of the mammalian immune system and an evolutionary highly conserved fundamental cellular process. A large number of molecules that are involved in phagocytosis are already known. However, a quantitative physical knowledge of this intrinsically mechanical process and therefore a comprehensive understanding is still lacking. Therefore, we investigated physical determinants of particle uptake and transport during phagocytosis. We examined the cellular resolution limit for particle uptake by using holographic optical tweezers in combination with correlative light and electron microscopy to measure the ability of cells to discriminate between two spatially separated objects. These studies provide insights into the spreading of cell signaling and the areas of signal integration during the uptake of particles. Using a combination of traction force microscopy and holographic optical tweezers, we were able to measure cellular forces during phagocytic uptake in a spatially and temporally resolved manner. The subsequent intracellular transport of phagosomes was investigated by live-cell particle tracking. We found that not all phagosomes are transported directly from the cell periphery to the perinuclear region. They rather exhibit more complex transport characteristics, which depend strongly on the size of the phagosomes. This transport behavior might be the foundation for a size-dependent cellular sorting mechanism for organelles. To gain insights into the underlying causes for this process, we quantified how the intracellular transport forces scale with the organelle size by using magnetic tweezers. These measurements provide cues for the numbers of participating motors and their potential cooperative behavior during the transport of different-sized organelles. In summary, we anticipate our results to pave the way for a deeper quantitative understanding of phagocytosis and thus enable the development of new and more comprehensive models for this important cellular process.

Rab32 GTPase, as a direct target of miR-30b/c, controls the intracellular survival of Burkholderia pseudomallei by regulating phagosome maturation.

Burkholderia pseudomallei is a gram-negative, facultative intracellular bacterium, which causes a disease known as melioidosis. Professional phagocytes represent a crucial first line of innate defense against invading pathogens. Uptake of pathogens by these cells involves the formation of a phagosome that matures by fusing with early and late endocytic vesicles, resulting in killing of ingested microbes. Host Rab GTPases are central regulators of vesicular trafficking following pathogen phagocytosis. However, it is unclear how Rab GTPases interact with B. pseudomallei to regulate the transport and maturation of bacterial-containing phagosomes. Here, we showed that the host Rab32 plays an important role in mediating antimicrobial activity by promoting phagosome maturation at an early phase of infection with B. pseudomallei. And we demonstrated that the expression level of Rab32 is increased through the downregulation of the synthesis of miR-30b/30c in B. pseudomallei infected macrophages. Subsequently, we showed that B. pseudomallei resides temporarily in Rab32-positive compartments with late endocytic features. And Rab32 enhances phagosome acidification and promotes the fusion of B. pseudomallei-containing phagosomes with lysosomes to activate cathepsin D, resulting in restricted intracellular growth of B. pseudomallei. Additionally, Rab32 mediates phagosome maturation depending on its guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding state. Finally, we report the previously unrecognized role of miR-30b/30c in regulating B. pseudomallei-containing phagosome maturation by targeting Rab32 in macrophages. Altogether, we provide a novel insight into the host immune-regulated cellular pathway against B. pseudomallei infection is partially dependent on Rab32 trafficking pathway, which regulates phagosome maturation and enhances the killing of this bacterium in macrophages.

Size-Dependent Organelle Transport during Phagocytosis

Phagocytosis of bacteria and other pathogens by macrophages is a key process of the mammalian immune system. The intracellular maturation of phagosomes which often leads to the degradation of the internalized pathogens shows high organelle-to-organelle variations that are not clearly understood. An important part of the maturation is the phagosomal transport from the cell periphery towards the perinuclear region. We hypothesize that the phagosome size influences the phagosomal transport and therefore also potentially the maturation process. We tested this hypothesis by tracking phagosomes with different diameters between 1 µm and 3 µm inside macrophages. We show that the transport efficiency increases with increasing phagosome size although the instantaneous velocities of the investigated phagosomes are very similar to each other. In addition, we found that the bi-directional motion as well as the transport from the nucleus back to the periphery decreases with increasing phagosome size. We furthermore show that dynein is significantly involved in the phagosomal transport, in particular in the persistent centripetal transport of large phagosomes. In addition, we found that actin-dependent motion is also contributing to the transport, in particular to the transport of small phagosomes. Furthermore we investigated the spatial distribution of dyneins and microtubules, and found that density differences between the nucleus-facing side of phagosomes and the opposite side can explain part of the observed transport characteristics. Our findings suggest that a basic size-dependent cellular sorting mechanism might exist that supports inward transport of large phagocytosed pathogens for facilitating their digestion and that simultaneously supports outward transport of small pathogen fragments for example for antigen presentation.

Actin nucleator Spire 1 is a regulator of ectoplasmic specialization in the testis

Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell–cell and Sertoli cell–spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood–testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell–cell interface, and the apical ES at the Sertoli–spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.

Phagosomal transport depends strongly on phagosome size

Macrophages internalize pathogens for intracellular degradation. An important part of this process is the phagosomal transport from the cell periphery to the perinuclear region. Biochemical factors are known to influence the fate of phagosomes. Here, we show that the size of phagosomes also has a strong influence on their transport. We found that large phagosomes are transported persistently to the nucleus, whereas small phagosomes show strong bidirectional transport. We show that dynein motors play a larger role in the transport of large phagosomes, whereas actin filament-based motility plays a larger role in the transport of small phagosomes. Furthermore, we investigated the spatial distribution of dyneins and microtubules around phagosomes and hypothesize that dynein and microtubule density differences between the nucleus-facing side of phagosomes and the opposite side could explain part of the observed transport characteristics. Our findings suggest that a size-dependent cellular sorting mechanism might exist that supports macrophages in their immunological roles.

The Voltage-Dependent Anion Channels (VDAC) of Mycobacterium avium phagosome are associated with bacterial survival and lipid export in macrophages

Mycobacterium avium subsp. hominissuis is associated with infection of immunocompromised individuals as well as patients with chronic lung disease. M. avium infects macrophages and actively interfere with the host killing machinery such as apoptosis and autophagy. Bacteria alter the normal endosomal trafficking, prevent the maturation of phagosomes and modify many signaling pathways inside of the macrophage by secreting effector molecules into the cytoplasm. To investigate whether M. avium needs to attach to the internal surface of the vacuole membrane before releasing efferent molecules, vacuole membrane proteins were purified and binding to the surface molecules present in intracellular bacteria was evaluated. The voltage-dependent anion channels (VDAC) were identified as components of M. avium vacuoles in macrophages. M. avium mmpL4 proteins were found to bind to VDAC-1 protein. The inactivation of VDAC-1 function either by pharmacological means or siRNA lead to significant decrease of M. avium survival. Although, we could not establish a role of VDAC channels in the transport of known secreted M. avium proteins, we demonstrated that the porin channels are associated with the export of bacterial cell wall lipids outside of vacuole. Suppression of the host phagosomal transport systems and the pathogen transporter may serve as therapeutic targets for infectious diseases.

Phagosomes of Different Size Show Qualitatively Different Transport Characteristics

As one of the key processes during the immune response, phagocytosis of bacteria plays a significant role in the mammalian immune system. A crucial part of phagocytosis is the transport of phagosomes from the cell periphery to the perinuclear region. This maturation process shows high variations from phagosome to phagosome that are not clearly understood. We hypothesize that the phagosome size has an influence on the maturation process by directly influencing the transport characteristics. We tested this hypothesis by tracking the transport of IgG-coupled microspheres with different diameters ranging from 1 µm to 3 µm inside macrophages. We show that phagosomal transport efficiency increases with increasing phagosome size despite nearly equal instantaneous velocities for all sizes. Furthermore, we show that the bi-directional intracellular motion of phagosomes as well as the transport from perinuclear region back to the periphery decreases with increasing phagosome size. Resting upon the notion that long range phagosomal transport is based on microtuble associated motor proteins, we investigated the spatial distribution of microtubules. We found density differences between the nucleus-facing side of phagosomes and the opposite side, which could explain part of the observed transport characteristics. In addition, we tested the influence of microtubule-associated motor proteins on the transport by labelling or inhibiting motors in vivo. We found that the concentration of dyneins is increased in the perinuclear region compared to the cell periphery. Furthermore, we found that the motility of large phagosomes is strongly decreased in cells without active dynein. We suggest that a size-dependent cellular sorting mechanism might exist, which supports inward transport of large phagosomes containing complete pathogens for enhancing their digestion and which simultaneously supports transport of digested products of phagosomal content back to cell periphery for example for antigen presentation.

Investigating Actin Mechanics during Phagocytic Uptake and Transport

Phagocytosis, the internalization and consecutive digestion of biological material by macrophages is a major part of the innate mammalian immune response. Fc gamma receptor-mediated uptake is driven by actin recruitment to the internalization site and consecutive formation of membrane protrusions around the target object. Molecular motors and cytoskeletal elements contribute to the subsequent transport of phagosomes inside the cells. Although a large number of molecules that are involved in the phagocytic uptake and the phagosomal transport are identified by now, the mechanics of these processes are largely unknown. We therefore investigated the mechanics of phagocytic uptake and phagosomal transport by using live cell microscopy in combination with optical and magnetic tweezers with a focus on the role of actin filaments. We hypothesize that the actin recruitment during phagocytic uptake leads to a transient and localized increase of the stiffness of the uptake region.To test this hypothesis we induced Fc gamma receptor-mediated phagocytosis in J774A.1 mouse macrophages by offering the cells target microbeads coated with immunoglobulin-G. During the uptake we measured local cell stiffness changes with a ‘blinking optical traps’ technique executed via periodic intensity modulation of the optical force. Preliminary data indicates that that the stiffness of the uptake region increases temporarily during the uptake. In addition, we investigated the influence of actin filaments on the phagosomal transport by magnetic tweezers-based bead displacement assays and cytochalasin treatment of the cells.Our measurements are expected to identify characteristic length- and time-scales for the variation of cell mechanical properties during phagocytosis and to contribute to a more comprehensive understanding of this medically relevant process.

Intracellular Manipulation of Phagosomal Transport and Maturation Using Magnetic Tweezers.

Phagocytosis is an important process of the immune system by which pathogens are internalized and eliminated by phagocytic cells. Upon internalization, the phagosome matures and acidifies while being transported in a centripetal fashion. In this chapter, we describe protocols for simultaneous imaging of phagosomal acidification as well as their spatial manipulation by magnetic tweezers. First, we describe the protocols for functionalization of magnetic microbeads with pH-sensitive dyes and pH calibration of these particles. We also describe the preparation of magnetic tweezers and the calibration of forces that can be generated by these tweezers. We provide details of the design of the custom electrical and optical setup used for simultaneous imaging of phagosomal pH and phagosome's location. Finally, we provide a detailed description of the data analysis methodology.

Polarity protein Crumbs homolog-3 (CRB3) regulates ectoplasmic specialization dynamics through its action on F-actin organization in Sertoli cells.

Crumbs homolog 3 (or Crumbs3, CRB3) is a polarity protein expressed by Sertoli and germ cells at the basal compartment in the seminiferous epithelium. CRB3 also expressed at the blood-testis barrier (BTB), co-localized with F-actin, TJ proteins occludin/ZO-1 and basal ES (ectoplasmic specialization) proteins N-cadherin/β-catenin at stages IV-VII only. The binding partners of CRB3 in the testis were the branched actin polymerization protein Arp3, and the barbed end-capping and bundling protein Eps8, illustrating its possible role in actin organization. CRB3 knockdown (KD) by RNAi in Sertoli cells with an established tight junction (TJ)-permeability barrier perturbed the TJ-barrier via changes in the distribution of TJ- and basal ES-proteins at the cell-cell interface. These changes were the result of CRB3 KD-induced re-organization of actin microfilaments, in which actin microfilaments were truncated, and extensively branched, thereby destabilizing F-actin-based adhesion protein complexes at the BTB. Using Polyplus in vivo-jetPEI as a transfection medium with high efficiency for CRB3 KD in the testis, the CRB3 KD testes displayed defects in spermatid and phagosome transport, and also spermatid polarity due to a disruption of F-actin organization. In summary, CRB3 is an actin microfilament regulator, playing a pivotal role in organizing actin filament bundles at the ES.

Formin 1 Regulates Microtubule and F-Actin Organization to Support Spermatid Transport During Spermatogenesis in the Rat Testis.

Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.

Lipid Rafts Assemble Dynein Ensembles

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams.