Cytoplasmic Flow Research Articles

P-142 ICSI Semi Automation powered by AI: Gateway for Oocyte Quality Assessment real time based on biomechanics during ICSI

Abstract Study question Embryo selection based on oolemma elasticity and cytoplasm viscosity using semi automated CL-ICSI technology assisted by Artificial Intelligence Summary answer CL-ICSI syringe/technology is capable of converting embryologist visual and feel of each ICSI procedure into a measurable form for objectivity of assessment in real time. What is known already In last four decades, understanding and/or assessing oocyte quality (based on biomechanics) become one of the needs in order to define the embryo formation and selection. initial days of ICSI, embryologist identifies the oocyte pool as fragile, normal or hyper elastic groups based on visual and indentation methods. Further, a variety of advanced techniques were implemented such as ultra fast imaging, atomic force sensing, micro tactile sensors, microfluidics and zone modulus measuring methods. These techniques were implemented on oocyte donation programs, and limited to research, due to the complexity and technical difficulties in implementing. Study design, size, duration From our observations, we would like to introduce our innovative technology (CL-ICSI) that is semi automation of ICSI procedure which is capable of measuring oolemma elasticity and cytoplasmic viscosity. we show the performance using limited number of discarded oocytes as a proof of concept of our technology. We believe that, exploitation of such technology become an asset/gateway for better understanding of oocyte quality and perhaps contribute in embryo selection based on oocyte biomechanics/quality. Participants/materials, setting, methods Current technology (CL-ICSI) is based on ICSI syringes that are calculated/designed to improve objectivity during the ICSI procedure among inter and intra users. CL-ICSI technology comprises of a semi automated syringe equipped with micro controllers that utilises force sensing and pressure mapping techniques to convert oocyte biomechanics data into measurable read outs on display unit. These measurable readouts are finally fed to AI algorithms that calculate the oocyte quality score as an output. Main results and the role of chance CL-ICSI semi automation technology is designed to minimise embryologist subjectivity during the ICSI procedure. Existing syringes works on manual rotatory mechanism which does not provide the start and stop point of the pressure applied on oocyte for puncturing, which genratlly controlled by embryologists skill and experience. Current technology comes with a click button that is designed to be loaded with potential energy which is proportional to the pressure/force required to puncture oolemma subsequently for sperm deposition. In case of classical syringes, post puncturing opposite rotation is implemented to withdraw the negative pressure or cytoplasmic content flow into the needle. Due to the lack of precise positioning the system become subjective. For this reason, we introduced mechanical potential energy build up, where oolemma puncture occurs with a button click and compensation of negative pressure occurs with releasing the button. This mechanism provides objectivity as well requires less than half of the time for oocyte intervention during ICSI over conventional rotatory syringes. More over, this technology works on pneumatics and is adaptable to all existing micro manipulators. Utilisation of such technologies may help improve the objectivity during ICSI and help understand oocyte quality in addition to the visual morphology assessment using AI. Limitations, reasons for caution CL-ICSI technology may be considered as next generation ICSI systems. Application of this technology in clinical setting in collecting the data from multicenters and different ethnicities might provide information that is a step closer to understand the oocyte quality and its role in embryo formation using artificial intelligence. Wider implications of the findings Embryo selection is one of the key event for single embryo transfer for healthy pregnancies. Morphology and morphokinetics data on oocytes and embryos provide valuable information but are limited, if there are no embryos formed. Current technology (CL-ICSI) may provide new avenues for better understanding on embryo prediction and selection. Trial registration number `not applicable`

Study on the Mechanism of Calcium in Osmotic Cell Cytoplasmic Flow

Study on the Mechanism of Calcium in Osmotic Cell Cytoplasmic Flow

Cullin-5 mutants reveal collective sensing of the nucleocytoplasmic ratio in Drosophila embryogenesis

In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT).1 These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows.2,3 In shackleton (shkl) mutants, this axial spreading is impaired.4 Here, we show that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that we exploit to study cell-cycle control as a function of the N/C ratio. We found that the N/C ratio is sensed collectively in neighborhoods of about 100μm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, we found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, we reveal a new pathway controlling nuclear positioning and provide a dissection of how nuclear cyclesrespond to the N/C ratio.

New observations on test architecture and construction of Jullienella foetida Schlumberger, 1890, the largest shallow-water agglutinated foraminifer in modern oceans.

We present new observations on Jullienella foetida Schlumberger, 1890, a giant agglutinated foraminifer with a leaf- or fan-like test reaching a maximum dimension of 14 cm, that is common on some parts of the west African continental shelf. The test wall comprises a smooth, outer veneer of small (<10 µm) mineral grains that overlies the much thicker inner layer, which has a porous structure and is composed of grains measuring several hundreds of microns in size. Micro-CT scans suggest that much of the test interior is filled with cytoplasm, while X-ray micrographs reveal an elaborate system of radiating internal partitions that probably serve to channel cytoplasmic flow and strengthen the test. Jullienella foetida resembles some xenophyophores (giant deep-sea foraminifera) in terms of test size and morphology, but lacks their distinctive internal organization; the similarities are therefore likely to be convergent. Based on micro-CT scan data, we calculated an individual cytoplasmic biomass of 3.65 mg wet weight for one specimen. When combined with literature records of seafloor coverage, this yielded an estimate of >7.0 g wet weight m−2 for the seafloor biomass of J. foetida in areas where it is particularly abundant. The relatively restricted distribution of this species off the north-west African coast at depths above 100 m is probably related to the elevated, upwelling-related surface productivity along this margin, which provides enough food to sustain this high biomass. This remarkable species appears to play an important, perhaps keystone, role in benthic ecosystems where it is abundant, providing the only common hard substrate on which sessile organisms can settle.

Class I myosin mediated endocytosis and polarization growth is essential for pathogenicity of Magnaporthe oryzae.

In eukaryotes, myosin provides the necessary impetus for a series of physiological processes, including organelle movement, cytoplasmic flow, cell division, and mitosis. Previously, three members of myosin were identified in Magnaporthe oryzae, with class II and class V myosins playing important roles in intracellular transport, fungal growth, and pathogenicity. However, limited is known about the biological function of the class I myosin protein in the rice blast fungus. Here, we found that Momyo1 is highly expressed during conidiation and infection. Functional characterization of this gene via RNA interference (RNAi) revealed that Momyo1 is required for vegetative growth, conidiation, melanin pigmentation, and pathogenicity of M. oryzae. The Momyo1 knockdown mutant is defective in formation of appressorium-like structures (ALS) at the hyphal tips. In addition, Momyo1 also displays defects on cell wall integrity, hyphal hydrophobicity, extracellular enzyme activities, endocytosis, and formation of the Spitzenkörper. Furthermore, Momyo1 was identified to physically interact with the MoShe4, a She4p/Dim1p orthologue potentially involved in endocytosis, polarization of the actin cytoskeleton. Overall, our findings provide a novel insight into the regulatory mechanism of Momyo1 that is involved in fungal growth, cell wall integrity, endocytosis, and virulence of M. oryzae. KEY POINTS: • Momyo1 is required for vegetative growth and pigmentation of M. oryzae. • Momyo1 is essential for cell wall integrity and endocytosis of M. oryzae. • Momyo1 is involved in hyphal surface hydrophobicity of M. oryzae.

Effects of chloroplast-cytoplasm exchange and lateral mass transfer on slow induction of chlorophyll fluorescence in Characeae.

Rapid cytoplasmic streaming in characean algae mediates communications between remote cell regions exposed to uneven irradiance. The metabolites exported from brightly illuminated chloroplasts spread along the internode with the liquid flow and cause transient changes in chlorophyll fluorescence at cell areas that are exposed to dim light or placed shortly in darkness. The largest distance to which the photometabolites can be transported has not yet been determined. Neither is it known if lateral transport has an influence on the induction of chlorophyll fluorescence. In this study, the relations between spatial connectivity of anchored chloroplasts in characean internodes and fluorescence induction curves were examined. Connectivity between remote cell parts was pronounced upon illumination of a cell spot at a distance up to 10 mm from the area of fluorescence measurement, provided the spot was located upstream in the cytoplasmic flow. Spatial interactions between distant cell sites were also manifested in strikingly different slow stages of fluorescence induction caused by narrow- and wide-field illumination. Cytochalasin D, cooling of bath solution, and inactivation of light-dependent envelope transporters were used to disturb cyclosis-mediated spatial interactions. Although fluorescence induction curves induced by narrow- and wide-field illumination differed greatly under control conditions, they became similar after the inhibition of cyclosis with cytochalasin D. The results indicate that cytoplasmic streaming not only drives the lateral translocation of photometabolites but also promotes the export of reducing power from illuminated chloroplasts due to flushing the chloroplast surface and keeping sharp concentration gradients.

Cytoplasmic streaming in C. elegans: Forces that drive oogenesis

In the gonad of C. elegans worms, germ cells form the outer lining of the central tube, the rachis, which carries a cytoplasmic flow. The porous interface between the germ cells and rachis allows a radial flow of cytoplasm across the interface. In the upstream, the flow at the interface is radially inwards boosting the main cytoplasmic flow in the rachis, while in the downstream the flow is radially outward feeding the germ cells to become oocytes. We analyze the cytoplasmic flow in the rachis as a flow in a porous tube of varying width, with spatially heterogeneous flow injection or leakage rates. Taking the geometry of the rachis into account, we predict properties of the rachis flow, such as pressure gradients and spatially heterogeneous flow speeds. Based on these, we speculate how directions of sub-flows between germ cells and rachis could be regulated for the development of healthy oocytes. Knowledge of this connection between micro-scale flow mechanics and physiological parameters may then also help us understand the abnormalities of reproduction in C. elegans worms with mutations or pathologies.

Cell cycle control during early embryogenesis.

Understanding the mechanisms of embryonic cell cycles is a central goal of developmental biology, as the regulation of the cell cycle must be closely coordinated with other events during early embryogenesis. Quantitative imaging approaches have recently begun to reveal how the cell cycle oscillator is controlled in space and time, and how it is integrated with mechanical signals to drive morphogenesis. Here, we discuss how the Drosophila embryo has served as an excellent model for addressing the molecular and physical mechanisms of embryonic cell cycles, with comparisons to other model systems to highlight conserved and species-specific mechanisms. We describe how the rapid cleavage divisions characteristic of most metazoan embryos require chemical waves and cytoplasmic flows to coordinate morphogenesis across the large expanse of the embryo. We also outline how, in the late cleavage divisions, the cell cycle is inter-regulated with the activation of gene expression to ensure a reliable maternal-to-zygotic transition. Finally, we discuss how precise transcriptional regulation of the timing of mitosis ensures that tissue morphogenesis and cell proliferation are tightly controlled during gastrulation.

Phylogenomics of Fargesia and Yushania reveals a history of reticulate evolution

AbstractReticulate evolution is a common and important driving force in angiosperm evolution. In this study, we analyzed the phylogenetic signals of genomic regions with different inheritance patterns to understand the evolutionary process of organisms using species‐rich Himalaya–Hengduan taxa of bamboos (Fargesia Franchet and Yushania Keng). We constructed phylogenetic trees using different sampling strategies and reconstruction methods based on genome skimming and double digest restriction‐site‐associated DNA sequencing data. We assessed the congruence of topologies generated from different datasets and employed several approaches to reveal the causes of phylogenetic incongruence, including the detection of hybridization and introgression using PhyloNetworks and the D‐statistic test (ABBA‐BABA test). We found that, in the plastome‐based phylogeny, Fargesia bamboos can be clustered into three groups and Yushania was nested within one of them, which contradicts the nuclear–double digest restriction‐site‐associated DNA sequencing‐based phylogeny. Moreover, the genetic variation of chloroplast DNA is significantly correlated with geographical distribution. The strong signal of incomplete lineage sorting, hybridization, introgression, and cytoplasmic gene flow found among genera and species suggests that reticulate evolution is the main cause for the phylogenetic incongruence between nuclear and chloroplast datasets. Our results add evidence that genomes with different inheritance patterns can reveal distinct evolutionary histories of species and suggest that reticulate evolution is prevalent in rapidly diversifying groups.

Induction Changes of Chlorophyll Fluorescence in Chara Cells Related to Metabolite Exchange between Chloroplasts and Cytoplasmic Flow

Induction Changes of Chlorophyll Fluorescence in Chara Cells Related to Metabolite Exchange between Chloroplasts and Cytoplasmic Flow

Encoding memory in tube diameter hierarchy of living flow network

The concept of memory is traditionally associated with organisms possessing a nervous system. However, even very simple organisms store information about past experiences to thrive in a complex environment-successfully exploiting nutrient sources, avoiding danger, and warding off predators. How can simple organisms encode information about their environment? We here follow how the giant unicellular slime mold Physarum polycephalum responds to a nutrient source. We find that the network-like body plan of the organism itself serves to encode the location of a nutrient source. The organism entirely consists of interlaced tubes of varying diameters. Now, we observe that these tubes grow and shrink in diameter in response to a nutrient source, thereby imprinting the nutrient's location in the tube diameter hierarchy. Combining theoretical model and experimental data, we reveal how memory is encoded: a nutrient source locally releases a softening agent that gets transported by the cytoplasmic flows within the tubular network. Tubes receiving a lot of softening agent grow in diameter at the expense of other tubes shrinking. Thereby, the tubes' capacities for flow-based transport get permanently upgraded toward the nutrient location, redirecting future decisions and migration. This demonstrates that nutrient location is stored in and retrieved from the networks' tube diameter hierarchy. Our findings explain how network-forming organisms like slime molds and fungi thrive in complex environments. We here identify a flow networks' version of associative memory-very likely of relevance for the plethora of living flow networks as well as for bioinspired design.

Quantifying Intracellular Particle Flows by DIC Object Tracking

Label-free imaging techniques such as differential interference contrast (DIC) allow the observation of cells and large subcellular structures in their native, unperturbed states with minimal exposure to light. The development of robust computational image-analysis routines is vital to quantitative label-free imaging. The reliability of quantitative analysis of time-series microscopy data based on single-particle tracking relies on accurately detecting objects as distinct from the background, i.e., segmentation. Typical approaches to segmenting DIC images either involve converting images to those resembling phase contrast, mimicking the optics of DIC object formation, or using the morphological properties of objects. Here, we describe MATLAB based, single-particle tracking tool with a GUI for mobility analysis of objects from in vitro and in vivo DIC time-series microscopy. The tool integrates contrast enhancement with multiple modified Gaussian filters, automated threshold detection for segmentation and minimal distance-based two-dimensional single-particle tracking. We compare the relative performance of multiple filters and demonstrate the utility of the tool for DIC object tracking (DICOT). We quantify subcellular dynamics of a time series of Caenorhabditis elegans embryos in the one-celled stage by detecting birefringent yolk granules in the cytoplasm with high precision. The resulting two-dimensional map of oscillatory dynamics of granules quantifies the cytoplasmic flows driven by anaphasic spindle oscillations. The frequency of oscillations across the anterior-posterior (A-P) and transverse axes of the embryo correspond well with the reported frequency of spindle oscillations. We validate the quantitative accuracy of our method by tracking the in vitro diffusive mobility of micron-sized beads in glycerol solutions. Estimates of the diffusion coefficients of the granules are used to measure the viscosity of a dilution series of glycerol. Thus, our computational method is likely to be useful for both intracellular mobility and in vitro microrheology.

Fungal Automata

We study a cellular automaton (CA) model of information dynamics on a single hypha of a fungal mycelium. Such a filament is divided in compartments (here also called cells) by septa. These septa are invaginations of the cell wall and their pores allow for the flow of cytoplasm between compartments and hyphae. The septal pores of the fungal phylum of the Ascomycota can be closed by organelles called Woronin bodies. Septal closure is increased when the septa become older and when exposed to stress conditions. Thus, Woronin bodies act as informational flow valves. The one-dimensional fungal automaton is a binary-state ternary neighborhood CA, where every compartment follows one of the elementary cellular automaton (ECA) rules if its pores are open and either remains in state 0 (first species of fungal automata) or its previous state (second species of fungal automata) if its pores are closed. The Woronin bodies closing the pores are also governed by ECA rules. We analyze a structure of the composition space of cell-state transition and pore-state transition rules and the complexity of fungal automata with just a few Woronin bodies, and exemplify several important local events in the automaton dynamics.

Getting around the cell: physical transport in the intracellular world

Eukaryotic cells face the challenging task of transporting a variety of particles through the complex intracellular milieu in order to deliver, distribute, and mix the many components that support cell function. In this review, we explore the biological objectives and physical mechanisms of intracellular transport. Our focus is on cytoplasmic and intra-organelle transport at the whole-cell scale. We outline several key biological functions that depend on physically transporting components across the cell, including the delivery of secreted proteins, support of cell growth and repair, propagation of intracellular signals, establishment of organelle contacts, and spatial organization of metabolic gradients. We then review the three primary physical modes of transport in eukaryotic cells: diffusive motion, motor-driven transport, and advection by cytoplasmic flow. For each mechanism, we identify the main factors that determine speed and directionality. We also highlight the efficiency of each transport mode in fulfilling various key objectives of transport, such as particle mixing, directed delivery, and rapid target search. Taken together, the interplay of diffusion, molecular motors, and flows supports the intracellular transport needs that underlie a broad variety of biological phenomena.

Subcellular structure and behaviour in fungal hyphae.

This work briefly surveys the diversity of selected subcellular characteristics in hyphal tip cells of the fungal kingdom (Mycota). Hyphae are filamentous cells that grow by tip extension. It is a highly polarised mechanism that requires a robust secretory system for the delivery of materials (e.g. membrane, proteins, cell wall materials) to sites of cell growth. These events result it the self-assembly of a Spitzenkörper (Spk), found most often in the Basidiomycota, Ascomycota, and Blastocladiomycota, or an apical vesicle crescent (AVC), present in the most Mucoromycota and Zoopagomycota. The Spk is a complex apical body composed of secretory vesicles, cytoskeletal elements, and signaling proteins. The AVC appears less complex, though little is known of its composition other than secretory vesicles. Both bodies influence hyphal growth and morphogenesis. Other factors such as cytoskeletal functions, endocytosis, cytoplasmic flow, and turgor pressure are also important in sustaining hyphal growth. Clarifying subcellular structures, functions, and behaviours through bioimagining analysis are providing a better understanding of the cell biology and phylogenetic relationships of fungi. LAY DESCRIPTION: Fungi are most familiar to the public as yeast, molds, and mushrooms. They are eukaryotic organisms that inhabit diverse ecological niches around the world and are critical to the health of ecosystems performing roles in decomposition of organic matter and nutrient recycling (Heath, 1990). Fungi are heterotrophs, unlike plants, and comprise the most successful and diverse phyla of eukaryotic microbes, interacting with all other forms of life in associations that range from beneficial (e.g., mycorrhizae) to antagonistic (e.g., pathogens). Some fungi can be parasitic or pathogenic on plants (e.g., Cryphonectria parasitica, Magnaporthe grisea), insects (e.g., Beauveria bassiana, Cordyceps sp.), invertebrates (e.g., Drechslerella anchonia), vertebrates (e.g., Coccidioides immitis, Candia albicans) and other fungi (e.g., Trichoderma viride, Ampelomyces quisqualis). The majority of fungi, however, are saprophytes, obtaining nutrition through the brake down of non-living organic matter.

Flow Induced Symmetry Breaking in a Conceptual Polarity Model.

Important cellular processes, such as cell motility and cell division, are coordinated by cell polarity, which is determined by the non-uniform distribution of certain proteins. Such protein patterns form via an interplay of protein reactions and protein transport. Since Turing’s seminal work, the formation of protein patterns resulting from the interplay between reactions and diffusive transport has been widely studied. Over the last few years, increasing evidence shows that also advective transport, resulting from cytosolic and cortical flows, is present in many cells. However, it remains unclear how and whether these flows contribute to protein-pattern formation. To address this question, we use a minimal model that conserves the total protein mass to characterize the effects of cytosolic flow on pattern formation. Combining a linear stability analysis with numerical simulations, we find that membrane-bound protein patterns propagate against the direction of cytoplasmic flow with a speed that is maximal for intermediate flow speed. We show that the mechanism underlying this pattern propagation relies on a higher protein influx on the upstream side of the pattern compared to the downstream side. Furthermore, we find that cytosolic flow can change the membrane pattern qualitatively from a peak pattern to a mesa pattern. Finally, our study shows that a non-uniform flow profile can induce pattern formation by triggering a regional lateral instability.

Computational universality of fungal sandpile automata

Hyphae within the mycelia of the ascomycetous fungi are compartmentalised by septa. Each septum has a pore that allows for inter-compartmental and inter-hyphal streaming of cytosol and even organelles. The compartments, however, have special organelles, Woronin bodies, that can plug the pores. When the pores are blocked, no flow of cytoplasm takes place. Inspired by the controllable compartmentalisation within the mycelium of the ascomycetous fungi we designed two-dimensional fungal automata. A fungal automaton is a cellular automaton where communication between neighbouring cells can be blocked on demand. We demonstrate computational universality of the fungal automata by implementing sandpile cellular automata circuits there. We reduce the Monotone Circuit Value Problem to the Fungal Automaton Prediction Problem. We construct families of wires, cross-overs and gates to prove that the fungal automata are P-complete.

On Boolean gates in fungal colony

A fungal colony maintains its integrity via flow of cytoplasm along mycelium network. This flow, together with possible coordination of mycelium tips propagation, is controlled by calcium waves and associated waves of electrical potential changes. We propose that these excitation waves can be employed to implement a computation in the mycelium networks. We use FitzHugh–Nagumo model to imitate propagation of excitation in a single colony of Aspergillus niger. Boolean values are encoded by spikes of extracellular potential. We represent binary inputs by electrical impulses on a pair of selected electrodes and we record responses of the colony from sixteen electrodes. We derive sets of two-inputs-on-output logical gates implementable the fungal colony and analyse distributions of the gates.

The maternal coordinate system: Molecular-genetics of embryonic axis formation and patterning in the zebrafish.

Axis specification of the zebrafish embryo begins during oogenesis and relies on proper formation of well-defined cytoplasmic domains within the oocyte. Upon fertilization, maternally-regulated cytoplasmic flow and repositioning of dorsal determinants establish the coordinate system that will build the structure and developmental body plan of the embryo. Failure of specific genes that regulate the embryonic coordinate system leads to catastrophic loss of body structures. Here, we review the genetic principles of axis formation and discuss how maternal factors orchestrate axis patterning during zebrafish early embryogenesis. We focus on the molecular identity and functional contribution of genes controlling critical aspects of oogenesis, egg activation, blastula, and gastrula stages. We examine how polarized cytoplasmic domains form in the oocyte, which set off downstream events such as animal-vegetal polarity and germ line development. After gametes interact and form the zygote, cytoplasmic segregation drives the animal-directed reorganization of maternal determinants through calcium- and cell cycle-dependent signals. We also summarize how maternal genes control dorsoventral, anterior-posterior, mesendodermal, and left-right cell fate specification and how signaling pathways pattern these axes and tissues during early development to instruct the three-dimensional body plan. Advances in reverse genetics and phenotyping approaches in the zebrafish model are revealing positional patterning signatures at the single-cell level, thus enhancing our understanding of genotype-phenotype interactions in axis formation. Our emphasis is on the genetic interrogation of novel and specific maternal regulatory mechanisms of axis specification in the zebrafish.

Hydrodynamic interactions of filaments polymerizing against obstacles.

Polymerization and depolymerization of cytoskeletal filaments against cellular structures can generate forces that are key to many cellular processes, such as cell motility and chromosomes movements during cell division. Motions generated by these forces induce global cytoplasmic flows and couple the dynamics of the polymerizing filaments and other bodies immersed in the fluid through their long-range hydrodynamic interactions (HIs). Previous theoretical and computational studies have largely ignored HIs. We use three dimensional discrete simulations to study the relationship between polymerization forces and their resulting flows and HIs. As a model system, we choose a filament that is polymerizing against an obstacle, and is embedded in a cylindrical array of parallel filaments of the same length. We consider three distinct mechanical scenarios for the filaments within the array: (a) all of the filaments are polymerizing with the same velocity; (b) they are all fixed in space, and (c) they are freely suspended. We show that each of these conditions produce their unique cytoplasmic flows and each result in differentiable polymerization forces and velocities. We also study the effect of buckling of filaments on polymerization forces and velocities and discuss the effect of HIs on the onset of buckling transition. Finally, we show that HIs result in the bundling of the buckled filaments within the array.