Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process of three human-infecting microsporidian species in vitro: Anncaliia algerae, Encephalitozoon hellem and E. intestinalis. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm�s-1 , and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is at least 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

Microsporidia, a divergent group of single-celled eukaryotic parasites, harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host. The organization of this specialized infection apparatus in the spore, how it is deployed, and how the nucleus and other large cargo are transported through the narrow PT are not well understood. Here we use serial block-face scanning electron microscopy to reveal the 3-dimensional architecture of the PT and its relative spatial orientation to other organelles within the spore. Using high-speed optical microscopy, we also capture and quantify the entire PT germination process in vitro. Our results show that the emerging PT experiences very high accelerating forces to reach velocities exceeding 300 μm⋅s -1 , and that firing kinetics differ markedly between species. Live-cell imaging reveals that the nucleus, which is approximately 7 times larger than the diameter of the PT, undergoes extreme deformation to fit through the narrow tube, and moves at speeds comparable to PT extension. Our study sheds new light on the 3-dimensional organization, dynamics, and mechanism of PT extrusion, and shows how infectious cargo moves through the tube to initiate infection.

Intracellular pathogens utilize various mechanisms in order to gain entry into host cells. Microsporidia are obligate intracellular, spore-forming parasites that harness a unique and specialized harpoon-like invasion organelle called the polar tube to initiate infection. In a dormant spore, the polar tube is packaged within the spore. Under suitable environments such as high pH and high ionic concentrations, the polar tube shoots out of the spore as a linear tube, which the length of the tube can be approximately 30 times of its spore size. The polar tube is thought to penetrate the host cell membrane and serve as a conduit to transfer infectious cargo into the host. Microsporidian polar tube firing is one of the fastest known biological processes, and has not been well characterized. In this study, we address how the polar tube is packaged within an intact spore, the kinetics of polar tube firing in different species, and how the infectious cargo is translocated. We used serial block-face scanning electron microscopy to reveal the three-dimensional architecture of the polar tube and its relative spatial organization to other organelles. The polar tube is a right-handed coil, and is tilted relative to the anterior-posterior axis of spores. We optimized a high-speed, wide-field optical microscopy to capture and quantify the entire polar tube firing process in vitro. Our results indicate that polar tube firing kinetics differ for different species. Maximum velocity and acceleration reach approximately 300 μm/s and 6,600 μm/s 2 , respectively. As polar tube firing happens, cargo begins to travel through the polar tube. We used nucleus as a marker and observed that the nucleus, which is many times the size of the polar tube diameter, undergoes vast deformation to fit into the narrow polar tube, and moves at speeds faster than polar tube firing. Our study sheds new lights on the structure, dynamics of the polar tube firing, and unravels how cargo moves through the narrow tube.

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