File Name: lysosome structure and function .zip
A simple description of lysosomes is that they are tiny sacs filled with fluid containing enzymes i. A lysosome is a type of membrane-bound organelle that is present in animal cells. Ranging in diameter from approx.
When food is eaten or absorbed by the cell, the lysosome releases its enzymes to break down complex molecules including sugars and proteins into usable energy needed by the cell to survive. In addition to their role as the digestive component and organelle-recycling facility of animal cells, lysosomes are considered to be parts of the endomembrane system. Lysosomes also use their hydrolytic enzymes to destroy pathogens disease-causing organisms that might enter the cell. In a process known as phagocytosis or endocytosis, a section of the plasma membrane of the macrophage invaginates folds in and engulfs a pathogen.
Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport.
We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking.
We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Lysosomes are membrane-bound organelles essential for endocytosis, phagocytosis, and autophagy  — .
Fusion of endosomes, phagosomes, and autophagosomes with lysosomes exposes the cargo in these organelles to the hydrolytic enzymes and low pH of the lysosomes resulting in the degradation of cargo. Like many organelles, the diameters of lysosomes are heterogeneous. The canonical lysosome diameter ranges from 50 nm to nm . A fundamental question is how the size of the lysosome affects intracellular transport.
Lysosome mobility is a combination of active transport and diffusion  — . Active, ATP-dependent, transport is driven by motor proteins, kinesin and dynein, moving the lysosome along microtubules.
Lysosomes also undergo periods of diffusion. Diffusion can be free or constrained by cellular structures, leading to sub-diffusive behavior. Lysosome diffusion should scale inversely with the diameter of lysosomes, with the caveat that viscosity can vary as a function of sub-cellular environment or cell cycle  , . It is less clear how lysosome diameter affects active transport. It is possible that the viscosity of the cytosol results in a diameter-dependent drag force on the lysosome.
Previous results have shown that organelle transport varies as a function of number of motor proteins for peroxisomes in insect cells  , vesicles in model neurons  , and melanosomes in Xenopus cells .
These results suggest that cytosolic drag is an important factor in intracellular transport. However, conflicting results have been obtained showing the kinesin-mediated transport of lipid droplets in Drosophila embryos is independent of the number of motors .
Overall, the importance of cytosolic drag on intracellular transport has been controversial  — . Our goal was to determine how lysosome diameter affects lysosome transport within live cells. In order to measure the transport of lysosomes as a function of diameter, we increased the diameter of lysosomes by incubating cells with sucrose.
Sucrose accumulates in lysosomes, but is not degraded within the lysosomes, resulting in an osmotic swelling of the lysosome  — . Sucrose incubation resulted in a population of enlarged lysosomes with diameters above the diffraction limit that allowed us to measure lysosomal transport for small, untreated, lysosomes and enlarged, sucrose-swollen, lysosomes.
Normal and enlarged fluorescently-labeled lysosomes were tracked during intracellular transport using live cell fluorescence microscopy. Single particle tracking shows that, as expected, diffusion decreased for larger lysosomes. Active transport, both anterograde and retrograde, was not affected by the increased lysosome diameter. Understanding the intracellular transport of lysosomes is necessary to understand cellular function as well as the human diseases associated with lysosomal disruption or defects  ,  , .
In addition, this research addresses the broader question of how organelle size affects transport. Our results for lysosomes can be easily extended to other transport vesicles including early and late endosomes, clathrin-coated vesicles, and caveosomes as well as the viruses, nanoparticles, nutrients, and cell surface receptors that are transported within these vesicles. A Confocal fluorescence microscopy image of untreated BS-C-1 cells shows the normal cellular distribution and punctate appearance of lysosomes green labeled with EYFP.
The nuclei are stained with DAPI blue. B Incubation with sucrose 50 mM, 12 h leads to enlargement of lysosomes. The increased diameter gives the lysosomes a circular appearance. The inset shows an expanded view of the region in the red box. Lysosomes were enlarged by incubating cells with sucrose 12 h, 50 mM , which leads to an osmotic swelling of the lysosomes Figure 1B  — . The increased diameter of the lysosomes is observed in fluorescence microscopy images as a population of circular, rather than punctate, lysosomes.
In addition to the increased diameter, a subset of the lysosomes form clusters after incubation with sucrose. Clustered lysosomes were not analyzed as the clusters themselves can inhibit motion. Experiments were also carried out in human cervix epithelial cells HeLa to ensure that results were not cell type dependent. A similar sucrose-dependent swelling of lysosomes was observed in HeLa cells Figure S1 , although a longer incubation period was required 24 h, 50 mM.
Lysosome diameter was measured using ImageJ to analyze confocal fluorescence microscopy images Figure 2. Untreated cells have punctate lysosomes with a median diameter of nm measured for 50 lysosomes in 3 cells Figure 2A.
The caveat associated with this measurement is that the diameter of the punctate lysosomes approaches the point spread function of the confocal microscope used for these experiments nm.
Circular lysosomes were not observed in untreated cells. In sucrose-treated cells, the circular lysosomes have a median diameter of 1. A Distribution of lysosome diameters measured in control, untreated cells. B Incubation with sucrose shifts the distribution of lysosome diameters to greater values. We also tested whether longer incubation times and higher concentrations of sucrose mM, 24 hrs would lead to even larger lysosomes data not shown.
We found that a greater percent of lysosomes were enlarged, but the median diameter remained the same. Lysosomal motion consists of active, ATP-dependent, transport along microtubules and periods of diffusion  — . This is observed in individual trajectories as stretches of long-range, directed motion interspersed with random, diffusive motion Figure 3.
Representative trajectories of 4 untreated lysosomes. Trajectories show periods of long-range transport and periods of diffusion. An image was recorded every 0. Images were recorded every 0. Free diffusion is characterized by a linear mean square displacement MSD with a slope of 4D. An analysis of lysosomes from 7 untreated cells shows that normal lysosomes diffuse with a broad range of diffusion coefficients Figure 4A. Averaging the MSDs of the lysosomes results in an average diffusion coefficient of 0.
In comparison, the diffusion coefficients of the enlarged lysosomes in sucrose-treated cells show a narrowed distribution shifted to lower values Figure 4B. The diffusion coefficient calculated from averaged MSDs decreases to 0. As sucrose-treatment does not enlarge all lysosomes, it was also possible to compare punctate and enlarged lysosomes, both within sucrose-treated cells, which showed a similar trend Figure S2. Similar results were obtained for lysosomes in HeLa cells Figure S3.
A Diffusion coefficients from punctate lysosomes in 7 untreated cells. B Diffusion coefficients from enlarged lysosomes in 7 sucrose-treated cells. Both MSD curves are fit to a line with a slope of 4D. Error bars show standard error. Active transport was defined as periods of directed lysosome motion in a single direction rather than the random motion associated with diffusion. Periods during which lysosomes moved in a single direction for a minimum of 1.
The velocity at each step was recorded. It is important to note that this measure of velocity is not that of a single motor protein step, but rather the distance traveled by the lysosome between frames. Lysosomes moving towards the cell periphery were classified as undergoing anterograde motion. For both control and sucrose-treated cells, 20 lysosomes in 5 cells were analyzed. Both control and sucrose-treated cells show a wide distribution of lysosome velocities Figure 5.
Histogram showing the distribution of lysosome velocities for punctate lysosomes in control cells solid gray and enlarged lysosomes in sucrose-treated cells black stripes cells. Data for each distribution was obtained from 20 lysosomes in 5 cells. The goal of this research was to determine how lysosome diameter affects lysosome transport. However, these were not all the same organelles. The largest organelles were mitochondria, the smaller organelles were not identified.
Incubating cells with sucrose results in enlarged lysosomes Figure 1 , allowing us to compare transport of identical organelles with different diameters within live cells. The increased diameter of the lysosomes, likely an osmotic effect  —  , gives them a circular appearance resulting from the LAMP1-EYFP fluorescent tag on the membrane of the lysosome. We use this difference in morphology to group lysosomes into two categories: punctate small and circular large.
While there are certainly larger punctate lysosomes and smaller circular lysosomes, this binning provides a method to separate lysosomes based on size during single particle tracking. Previous measurements of lysosome diameter range from 50 nm to nm . For example, mouse macrophage lysosomes have an average diameter of nm . Rat kidney fibroblasts have lysosomes with diameters of nm to nm .
Untreated, punctate, lysosomes in these monkey kidney epithelial cells have a median diameter of nm Figure 2A. This is likely an over-estimate as smaller lysosomes would be below the nm point spread function of the confocal microscope used to obtain images.
Enlarged, circular, lysosomes have median diameters of 1. The approximately 2-fold increase in diameter of lysosomes following sucrose incubation provides an ideal system for the comparison of identical organelles with different diameters.
The motion of lysosomes includes both active transport and passive diffusion Figure 3. The relationship between lysosome diameter and diffusive and active transport was examined by comparing the motion of punctate and enlarged lysosomes Figures 4 and 5.
Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking.
На крыше главного служебного здания АНБ вырос лес из более чем пятисот антенн, среди которых были две большие антенны, закрытые обтекателями, похожими на громадные мячи для гольфа. Само здание также было гигантских размеров - его площадь составляла более двух миллионов квадратных футов, вдвое больше площади штаб-квартиры ЦРУ. Внутри было протянуто восемь миллионов футов телефонного кабеля, общая площадь постоянно закрытых окон составляла восемьдесят тысяч квадратных футов. Сьюзан рассказала Дэвиду про КОМИ НТ, подразделение глобальной разведки, в распоряжении которого находилось немыслимое количество постов прослушивания, спутников-шпионов и подслушивающих устройств по всему земному шару. Ежедневно тысячи сообщений и разговоров перехватывались и посылались экспертам АНБ для дешифровки. Разведданные, поставляемые агентством, влияли на процесс принятия решений ФБР, ЦРУ, а также внешнеполитическими советниками правительства США. Беккер был потрясен.
Сьюзан отвернулась от экрана ВР к боковому монитору. На нем бесконечно повторялась видеозапись убийства Танкадо. И всякий раз Танкадо хватался за грудь, падал и с выражение ужаса на лице навязывал кольцо ничего не подозревающим туристам. В этом нет никакого смысла, - размышляла. - Если он не знал, что мы его убиваем… Ничего не понятно.
Я все рассказал лейтенанту. - Я с ним говорил, но… - Надеюсь, вы отчитали его как следует! - воскликнул Клушар. Беккер кивнул: - Самым решительным образом. Консульство этого так не оставит. - Надеюсь.
Они были вмонтированы так хитро, что никто, кроме Грега Хейла, их не заметил, и практически означали, что любой код, созданный с помощью Попрыгунчика, может быть взломан секретным паролем, известным только АНБ. Стратмору едва не удалось сделать предлагаемый стандарт шифрования величайшим достижением АНБ: если бы он был принят, у агентства появился бы ключ для взлома любого шифра в Америке. Люди, знающие толк в компьютерах, пришли в неистовство. Фонд электронных границ, воспользовавшись вспыхнувшим скандалом, поносил конгресс за проявленную наивность и назвал АНБ величайшей угрозой свободному миру со времен Гитлера.
Беккер шумно вздохнул. Разумеется. Но мне она неизвестна.
Где же .
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